CN115992273A - Nucleic acid molecule, kit and detection method for detecting streptococcus pneumoniae - Google Patents

Abstract

The invention discloses a CRISPR-based hands-free one-step detection technology (ExCad), and provides a nucleic acid molecule, a kit and a detection method for detecting streptococcus pneumoniae. The nucleic acid molecule for detecting streptococcus pneumoniae provided by the invention comprises an LAMP primer group, gRNA and ssDNA probes for detecting streptococcus pneumoniae. The nucleic acid molecule is matched with Cas12b protein, and a CRISPR-based one-step streptococcus pneumoniae detection method (ExCad-Sp) is further established by applying the ExCad technology, and a streptococcus pneumoniae detection kit is also established, and the kit has the advantages of low cost and simple and convenient operation, and can realize specific, sensitive, rapid and accurate streptococcus pneumoniae detection.

Description

Nucleic acid molecule, kit and detection method for detecting streptococcus pneumoniae

Technical Field

The invention relates to the technical field of nucleic acid detection, in particular to a CRISPR-based hands-free one-step detection technology (ExCad), and a nucleic acid molecule, a kit and a detection method for detecting streptococcus pneumoniae.

Background

Recently, emerging nucleic acid detection technologies based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) have been of great interest, and are known as one of seven technologies of interest in 2022. These CRISPR-based detection techniques are based on trans-cleavage (sidecut) activity of some CRISPR-associated (Cas) effector molecules, such as Cas12a, cas12b, cas13a and Cas14, and have been successfully applied to detect a variety of pathogens and small molecules. RNA-guided DNA-targeted Cas12 and RNA-guided RNA-targeted Cas13 have been used to develop novel nucleic acid detection platforms, e.g., DETECTR, SHERLOCK or HOLMES detection platforms. In these detection platforms, CRISPR-based detection is combined with nucleic acid amplification with high specificity and sensitivity.

However, up to now, on most CRISPR-mediated nucleic acid detection platforms, target pre-amplification and CRISPR-based detection are two independent steps, i.e. the reaction tube lid needs to be opened after pre-amplification, the pre-amplified product is manually transferred into the Cas detection system, and the next CRISPR detection is performed—this results in more cumbersome manual operations and potential cross-contamination risks. Some CRISPR-based detection platforms improve CRISPR-assisted single tube in-detection, but still require centrifugation and mixing steps. Furthermore, to ensure efficient operation of Cas enzymes, it is often necessary to place a Protospacer Adjacent Motif (PAM) sequence in the appropriate position, which is a limitation that prevents the application of CRISPR-based detection methods, as there may be no appropriate PAM sites in the target sequence. The CRISPR-top technology is a one-step one-pot CRISPR-based nucleic acid detection platform, combines loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12 b-based detection in one reaction tube, and avoids cross contamination to the greatest extent. In addition, in the CRISPR-top method, one standard PAM sequence of Cas12b is artificially inserted into the junction region of the Forward Inner Primer (FIP), and PAM sites are introduced into the target by LAMP pre-amplification, thereby expanding the application range of the technology. However, during CRISPR-top applications we found that in some cases it was difficult to design suitable FIP and guide RNAs (gRNA), especially for AT-rich sequences. In CRISPR-based assays, inappropriate FIP can lead to false positive results, presumably due to FIP folding itself and forming a gRNA-like structure that can activate Cas12 b. Furthermore, CRISPR-top analysis requires DNA/RNA extraction steps, which complicate the detection process and prevent the application of the method in laboratory-free conditions. Sputum, a non-invasive sample type, is readily available and is reliable in diagnosing respiratory disease. However, because of the complex components and high viscosity of sputum specimens, it is difficult to directly use the sputum specimens for nucleic acid detection without extracting nucleic acids.

Streptococcus pneumoniae (Streptococcus pneumoniae, sp) is the most predominant bacterial pathogen responsible for community-acquired pneumonia. Currently, clinical diagnosis of streptococcus pneumoniae is mainly dependent on traditional isolation culture techniques, which are still the gold standard for clinical diagnosis of streptococcus pneumoniae infection. However, the isolation and culture are time-consuming, require a certain clinical test experience and are not suitable for rapid detection and home self-test by means of mass spectrometers or gene amplification sequencing. Although the fluorescent quantitative PCR technique has been applied to the detection of Streptococcus pneumoniae and exhibits good effects, the fluorescent quantitative PCR instrument belongs to a precise instrument and is expensive, so that the fluorescent quantitative PCR method is difficult to realize not only in home self-test but also in other areas or scenes where conditions are limited. Although isothermal amplification methods do not rely on precision instruments, a number of studies have shown that detection using isothermal amplification alone is extremely prone to false positives, leading to misdiagnosis. Currently, methods for detecting streptococcus pneumoniae based on CRISPR have not been developed.

Disclosure of Invention

The invention aims to provide a nucleic acid molecule and a kit for detecting streptococcus pneumoniae, and a streptococcus pneumoniae detection method established by a hand-free one-step detection technology (ExCad) based on CRISPR.

In order to achieve the aim of the invention, the invention provides a rapid pretreatment process of a sputum specimen, which is simple, rapid and economic, can realize the liquefaction of the sputum specimen and release bacterial nucleic acid in the specimen only for 10min without special instruments and equipment, and is used for nucleic acid detection technologies based on PCR and isothermal amplification. The pretreatment method is used, a primer design scheme is optimized, a CRISPR-top technology is improved, a one-step CRISPR auxiliary detection platform without Extraction is designed, and the platform is named as ExCad (Extraction-free one-step CRISPR-assistant detection; CRISPR one-step detection is carried out in a hands-free mode) (figure 1). The 5' end of the LAMP loop primer is artificially added with a PAM sequence by ExCad, so that a more loose primer design scheme is provided for a CRISPR one-step detection technology, and a target sequence is accurately detected. Practice proves that the pure colony without extracting nucleic acid and the clinical sputum specimen subjected to rapid pretreatment can be directly used for ExCad, so that the detection workflow is greatly simplified, and the response time of a sample is shortened. The ExCad reaction is carried out at a constant temperature, and the detection result can be read by a fluorescence reader (such as a fluorescence quantitative PCR instrument, an enzyme-labeled instrument or other small-sized fluorescence reading equipment) or can be read by naked eyes under blue light.

In a first aspect, the present invention provides a LAMP primer group for detecting Streptococcus pneumoniae, comprising primers F3, B3, FIP, BIP, LF and LB, the nucleotide sequences of which are shown in SEQ ID NOS: 1-6, respectively.

In a second aspect, the present invention provides a set of nucleic acid molecules for detecting Streptococcus pneumoniae comprising the LAMP primer group (SEQ ID NOS: 1-6), gRNA and ssDNA probes.

Wherein, the gRNA targets the DNA fragment of streptococcus pneumoniae lytA gene, and the DNA fragment is obtained by adopting the LAMP primer group for amplification.

The ssDNA probe is single-stranded DNA conforming to the principle of trans-cleavage of Cas12b protein.

Preferably, the nucleotide sequence of the gRNA is shown in SEQ ID NO. 7.

Preferably, the nucleotide sequence of the ssDNA probe is shown as SEQ ID NO. 8, and the 5 'end and the 3' end of the ssDNA probe are respectively marked with a fluorescent group and a quenching group. Alternatively, the 5 'and 3' ends of the ssDNA probes are labeled with a fluorophore and biotin, respectively.

Specifically, when using a fluorescence-based result reading method, the 5 'end and 3' end of the ssDNA probe are labeled with a fluorescent group and a quenching group, respectively. When a lateral flow biosensor (i.e., CRISPR strip) is used as the result reading method, FAM/FITC and Biotin (Biotin) are labeled at the 5 'and 3' ends of the ssDNA probe, respectively.

In a third aspect, the invention provides a kit for detecting streptococcus pneumoniae, the kit comprising the nucleic acid molecule and a Cas12b protein.

Further, the kit further comprises at least one of LAMP reaction buffer, bst 2.0DNA polymerase, cas12b reaction buffer, and sputum digestate, spathasol.

In a fourth aspect, the invention provides a method for detecting streptococcus pneumoniae (including non-diagnostic purposes), using the kit to detect, comprising the steps of:

1) Preparation of Cas12b/gRNA complex: mixing Cas12b protein (preferably AapCas12b protein) and gRNA with Cas12b reaction buffer, and incubating at 35-39 ℃ for 5-15min (preferably incubating at 37 ℃ for 10 min) to obtain Cas12b/gRNA complex;

2) The LAMP primer group, bst 2.0DNA polymerase, LAMP reaction buffer, cas12b/gRNA complex, ssDNA probe and template are mixed and reacted at a constant temperature within the temperature range of 52-62 ℃ for at least 20min (preferably 57 ℃ for 1 h).

Further, before step 1), the method further comprises a step of preprocessing the sample, specifically as follows: adding 1-1.5 times volume of sputum digestion liquid Sputasol into a sample to be detected, incubating for 5-15min at 100 ℃ (preferably incubating for 10min at 100 ℃), and taking the pretreated sample as a template.

In the method, in the reaction system, the concentration of the primers F3 and B3 is 0.08-0.6 mu M, the concentration of the primers FIP and BIP is 0.34-2.38 mu M, the concentration of the primers LF and LB is 0.17-1.19 mu M, the concentration of the primers F3 and B3 is the same, the concentration of the primers FIP and BIP is the same, and the concentration of the primers LF and LB is the same.

Preferably, the concentration of primers F3, B3 is 0.17. Mu.M, the concentration of primers FIP, BIP is 0.68. Mu.M, and the concentration of primers LF and LB is 0.34. Mu.M.

The total volume of the system of the reaction was 25 μl, including: 12.5. Mu.L of 2 XLAP reaction buffer, 1. Mu.L of 8U Bst 2.0 enzyme, 0.34-2.38. Mu.L of each of primers FIP and BIP (preferably 0.68. Mu.M), 0.17-1.19. Mu.M of each of primers LF and LB (preferably 0.34. Mu.M), 0.08-0.6. Mu.M of each of primers F3 and B3 (preferably 0.17. Mu.M), 3-5. Mu.L of Cas12B/gRNA complex (preferably 3.5. Mu.L), 0.2-2.0. Mu.L of 100. Mu.M ssDNA probe (preferably 0.5. Mu.L), 1-5. Mu.L of template (preferably 1. Mu.L), and the concentration of primers F3 and B3 are the same, the concentration of primers FIP and BIP are the same, and the concentration of primers LF and LB are the same;

the preparation method of the Cas12b/gRNA complex comprises the following steps: 15pmol of Cas12b protein and 2. Mu.L of 10. Mu.M gRNA are placed in a Cas12b reaction buffer, and incubated at 35-39 ℃ for 5-15min (preferably at 37 ℃ for 10 min), thus obtaining the protein.

The sample to be tested may be sputum.

In a fifth aspect, the invention provides a nucleic acid molecule for detection of a target gene, the nucleic acid molecule comprising a LAMP primer set for detection of a target gene and a gRNA and ssDNA probe, and the nucleic acid molecule cooperating with a CRISPR-Cas protein being useful for detection of a target gene;

the LAMP primer group is designed according to a target gene, comprises an inner forward primer, an inner reverse primer, an outer forward primer, an outer reverse primer, a forward loop primer and/or a reverse loop primer, and modifies a PAM sequence at the 5' end of the loop primer;

the gRNA targets the DNA fragment of the target gene, and the DNA fragment is obtained by adopting the LAMP primer group for amplification;

the ssDNA probe is single-stranded DNA conforming to the principle of trans-cleavage of Cas proteins.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

the invention establishes a rapid pretreatment method of sputum samples for nucleic acid detection. The principle is as follows: dithiothreitol contained in sputum acts on disulfide bonds of mucin in sputum, so that the bonds are promoted to be broken, and the viscosity of the sputum is reduced; then heating at 100deg.C, bacterial cells in the sputum are ruptured, and nucleic acids are released.

(II) the invention establishes an ExCad detection technology by improving the one-step detection based on CRISPR, and the main improvement points are as follows: (1) By using the rapid pretreatment method of the sputum sample, the sample nucleic acid extraction step in the detection method based on CRISPR is eliminated, and the fact that bacteria without extracted nucleic acid can be directly used for CRISPR one-step detection is proved, so that the operation steps are obviously reduced, and the detection time is shortened; (2) the primer design scheme is further relaxed. The principle is as follows: an engineered loop primer is formed by artificially modifying the PAM sequence at the 5' end of the LAMP loop primer, and the PAM sequence is introduced into the amplified product by LAMP amplification. Therefore, when the ExCad primer is designed, the design is only carried out according to the LAMP primer design principle, and then the PAM sequence is artificially added at the 5' end of the loop primer. Thus, the method can detect any DNA sequence, whether or not the sequence contains PAM sites.

The accurate, rapid and sensitive one-step streptococcus pneumoniae detection method (ExCad-Sp) based on CRISPR is established by using the ExCad technology, the method has low cost and simple and convenient operation, and all reagents and consumables required by detection can be provided together with the kit, so that the method can be stably transported and stored. In addition, the detection method is independent of special equipment, and people without molecular biology base can easily and simply realize the nucleic acid self-detection of streptococcus pneumoniae at home according to the instructions. The kit is not only suitable for home self-nucleic acid detection of non-test experience personnel, but also suitable for community large-scale screening and other on-site rapid detection scenes. The method is also suitable for identifying strains without extracting nucleic acid.

Drawings

FIG. 1 is a workflow and reaction principle of the ExCad technology of the present invention. Wherein, a, the workflow of ExCad technology; b, reaction principle of ExCad technology.

FIG. 2 shows the primer and gRNA design of the ExCad-Sp detection method according to the preferred embodiment of the invention. Only a part of the nucleotide sequence (401-612 bp) of the sense strand of the lytA gene is shown. The right and left arrows represent the sense and complementary sequences used, respectively. Artificially added PAM Sequences (TTCs) are highlighted in a grey background. gRNA is boxed.

FIG. 3 shows the optimal reaction conditions for ExCad-Sp detection in a preferred embodiment of the invention. Wherein a, the optimal reaction temperature; b, optimal primer premix volume.

FIG. 4 shows the results of a specific analysis of ExCad-Sp detection in a preferred embodiment of the invention. Wherein a, using the DNA of different strains to detect ExCad-Sp; b, exCad-Sp detection was performed using a clinical isolate of Streptococcus pneumoniae strain from which DNA was not extracted. 1-40, a non-streptococcus pneumoniae strain; 41-61, streptococcus pneumoniae clinical isolates.

FIG. 5 shows the results of a sensitivity analysis of ExCad-Sp detection in a preferred embodiment of the invention. A, a real-time fluorescence value curve graph; error bars represent mean ± standard error; n=3 replicates; b, a bar graph of final fluorescence values after reaction; n=3 replicates, error bars represent mean ± standard error; adopting two-sided student t test; * P <0.0001; ns, no statistical significance; c, directly observing by naked eyes under blue light; and take a picture with a smart phone.

FIG. 6 is a comparison of 5 sputum specimen pretreatment methods in a preferred embodiment of the invention. Wherein, P1-P7 and P11-P13 are positive samples of streptococcus pneumoniae, and N1 is a negative sample of streptococcus pneumoniae.

FIG. 7 illustrates the feasibility of ExCad-Sp detection using rapidly pre-processed clinical samples in a preferred embodiment of the invention. Wherein, a-c, exCad-Sp detects the detection lower limit of the labeled sample. 10 ³ -10 ^-3 The number of streptococcus pneumoniae cells contained for each reaction; n=3 replicates, error bars represent mean ± standard error; adopting two-sided student t test; * P is:<0.01；****,P<0.0001; ns, no statistical significance. a, a real-time fluorescence value curve graph; b, a bar graph of final fluorescence values after reaction; and c, directly observing by naked eyes under blue light, and photographing by using a smart phone. d-f, exCad-Sp detects the effect of clinical sputum specimens. d, a scatter diagram. Error bars represent mean ± standard error of final fluorescence values after reaction; e, directly observing the final fluorescence value heat map after reaction under blue light by naked eyes and photographing by using a smart phone. False negative samples read with blue light are indicated by asterisks. P1-P14, streptococcus pneumoniae positive samples; N1-N18, streptococcus pneumoniae negative samples. f, agreement between mass spectrometry (culture-MS) and ExCad detection of 32 clinical sample results.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.

Sputum digest, sputasol, used in the examples below, was purchased from OXOID, UK and the AapCas12b protein (containing Cas12b reaction buffer) was purchased from Huidexin (Tianjin) biosciences.

Example 1ExCad technical design

The total volume of the ExCad reaction system was 25. Mu.L: 2 XLAP reaction buffer 12.5. Mu. L, bst 2.0.0 enzyme (8U) 1. Mu. L, FIP and BIP 0.68. Mu. M, LF each and LB 0.34. Mu. M, F3 each and B3 0.17. Mu. M, cas12B/gRNA complex 3.5. Mu. L, ssDNA probe 0.5. Mu.L (100. Mu.M), template (DNA or pre-treated sample) 1. Mu.L, and finally make up the total volume to 25. Mu.L using sterile Deionized Water (DW). The negative control used DW as template. When colonies on the plate were used directly for ExCad analysis, after the preparation of the reaction mixture was completed, colonies were picked up with a sterile pipette tip and mixed well with the mixture. The ExCad reaction was incubated at a constant temperature of 52-62℃for 1h, with different primer sets having different optimal temperatures. Finally, a fluorescence reader or a reaction tube is placed under blue light with the wavelength of 440-485 nm to read the result by naked eyes.

Cas12b/gRNA complexes can be prepared in advance: 15pmol of AapCas12b protein and 2. Mu.L of gRNA (10. Mu.M) were placed in Cas12b reaction buffer and incubated at 37℃for 10min. The prepared compound can be stored at 4 ℃ for 12 hours at most.

Example 2 detection of Streptococcus pneumoniae Using ExCad technology

To demonstrate the effectiveness of the ExCad technology, a detection method for detecting Streptococcus pneumoniae was established using the ExCad technology, and was named ExCad-Sp. The construction process of the method is as follows:

first, the internal primers F3 and B3 and the external primers FIP and BIP necessary for LAMP amplification were designed on-line by means of PrimerExplorer software for the species-specific gene-lytA sequence of Streptococcus pneumoniae (GenBank accession number: AP 018938.1). In designing the primer, the parameters such as Tm value, GC content, primer distance and the like are adjusted and optimized, in a plurality of primer combinations given by software, the primer groups targeting different DNA fragments are selected first, and the primer sequence screening and adjustment are carried out by combining the dG value of the primer, so that 15 sets of primers which can be well suitable for ExCad detection are finally obtained. Then, the design of the loop primer LF/LB was performed manually at the appropriate position of 15 sets of primers. According to the principle of ExCad detection, a standard PAM sequence TTC of Cas12b is added to the 5' end of each designed upstream loop primer LF or downstream loop primer LB. The LAMP primer and gRNA were synthesized and purified by HPLC. And (3) carrying out LAMP amplification on the 15 sets of primers under the same conditions, and selecting the primer group with highest amplification efficiency and best specificity. The method comprises the following steps:

a25. Mu.L LAMP reaction system was prepared, comprising 12.5. Mu.L of 2 XPAB reaction buffer, 1. Mu.L of Bst 2.0DNA polymerase (8U), 0.4. Mu.M primer F3/B3, 1.6. Mu.M primer FIP/BIP, 0.8. Mu.M primer LF/LB, 1. Mu.L template DNA, and finally the total volume was made up to 25. Mu.L using DW. Genomic DNA from Streptococcus pneumoniae Standard strain ATCC49619 was used as a positive control template, and sterile Deionized Water (DW) was used as a negative control template. The LAMP reaction was carried out at 59℃for 1 hour and the results were read with a real-time turbidimeter.

Finally, 1 set of primer group with highest amplification efficiency and best specificity is screened out, and gRNA is designed manually according to the amplification site and ExCad detection principle. The sequences of LAMP primers and gRNA are shown in Table 1, and the binding and amplification sites are shown in FIG. 2. Single-stranded DNA (ssDNA) probes were labeled with fluorescent/quenching groups, the sequences and labels of which are shown in Table 1.

TABLE 1 oligonucleotide sequences used in the present invention

Note that: ^a the artificially added PAM sequence (TTC) is indicated in bold.

Example 3 optimization of ExCad-Sp detection reaction conditions

First, the optimal reaction temperature for the ExCad-Sp assay was determined. The temperature range tested was 55-60℃with 1℃intervals. 3 replicates were run at each reaction temperature. As shown in FIG. 3a, the ExCad-Sp reaction efficiency is higher in the range of 56-58 ℃. In combination with the reaction time (better for shorter) and fluorescence value (better for higher), 57 ℃ was finally chosen as the optimal reaction temperature.

Then, the optimal working concentration of LAMP primer in ExCad-Sp detection was determined. The primers were diluted to 100. Mu.M with DW, 40. Mu.L each of FIP and BIP, 20. Mu.L each of LF and LB, and 10. Mu.L each of F3 and B3 were mixed to form a primer premix. Next, different reaction systems were prepared by premixing with different volumes (0.3-2.1. Mu.L) of primers and tested at 57 ℃.3 replicates were run for each reaction system. As shown in FIG. 3b, the reaction efficiency was highest at a primer premix concentration of 0.6. Mu.L. Thus, the optimal working concentration of LAMP primers in ExCad-Sp analysis was 0.68. Mu.M for FIP and BIP, 0.34. Mu.M for LF and LB, and 0.17. Mu.M for F3 and B3, respectively.

Example 4 sensitivity and specificity of ExCad-Sp detection method

To determine the specificity of the ExCad-Sp assay, we collected 21 clinical isolates of Streptococcus pneumoniae and 40 non-Streptococcus pneumoniae (Table 2), and extracted and purified DNA templates therefrom. The 21 clinical isolates of Streptococcus pneumoniae were also tested for non-DNA extraction. Genomic DNA and DW of Streptococcus pneumoniae reference strain ATCC49619 were used as positive and negative control templates, respectively. The results are shown in FIG. 4a, with all positive results from Streptococcus pneumoniae and all negative results from Streptococcus non-pneumoniae. The result read out under blue light is consistent with the result read out by the real-time fluorescence detector. False positive results were not seen in the experiment. Thus, our data indicate that ExCad-Sp detection has very strong specificity for the detection of Streptococcus pneumoniae. In addition, the ExCad-Sp detection using 21 Streptococcus pneumoniae clinical isolate colonies showed positive results and no significant difference in fluorescence values from the use of nucleic acids, indicating the feasibility of ExCad-Sp detection using colonies not extracting DNA (FIG. 4 b).

In the sensitivity test, genomic DNA extracted from ATCC49619 was quantified by a spectrophotometer and serially diluted from 1 ng/. Mu.L to 1 fg/. Mu.L at 10-fold intervals, and DNAs at different concentrations were used as templates for the sensitivity test, and DW was used as a template for the negative control. As a result, a significant increase in fluorescence value was observed from 1ng to 1pg of genomic DNA within 1 hour of the start of the reaction, whereas the fluorescence value from 100fg to 1fg of genomic DNA and the negative control did not significantly increase (FIGS. 5a and 5 b). Similar results were also obtained with the naked eye under blue light (fig. 5 c). Thus, the lower limit of detection of the present method is 1pg of bacterial genomic DNA.

TABLE 2 specific detection of strains used and ExCad-Sp detection results

Example 5 Rapid pretreatment method of sputum specimen

In order to explore a rapid pretreatment method of sputum specimens for ExCad detection, we tested 5 sputum specimens treatment methods, which are respectively: (1) untreated group: no treatment is performed; (2) NaOH group: adding 4% sodium hydroxide solution with the same volume as the sputum specimen, and fully and uniformly mixing until the sputum specimen is liquefied; (3) PBS group: adding 4% sodium hydroxide solution with the same volume as the sputum specimen, fully and uniformly mixing until the sputum specimen is liquefied, centrifuging to remove supernatant, re-suspending the sediment by using 1mL Phosphate Buffer (PBS), centrifuging again to remove supernatant, and finally re-suspending the sediment by using PBS with the same volume as the original sputum specimen; (4) Sputasol group: adding Sputasol sputum digestive juice with the same volume as the sputum specimen, and fully and uniformly mixing until the sputum specimen is liquefied; (5) Sputasol boiling group: adding Sputasol sputum digestive juice with the same volume as the sputum specimen, fully and uniformly mixing until the sputum specimen is liquefied, incubating for 10min at 100 ℃, and cooling to room temperature. We collected 10 streptococcus pneumoniae positive patient sputum specimens and tested the 5 treatments above against 1 streptococcus pneumoniae negative sputum specimen. The positive/negative results of the streptococcus pneumoniae are the identification results of separating culture and combining MALDI-TOF MS mass spectrometry (hereinafter referred to as mass spectrometry).

The test results for the 5 methods are as follows (fig. 6): (1) 50% of positive samples in the untreated group falsely showed negative results; (2) 100% of positive samples of NaOH groups displayed negative results erroneously; (3) 20% of positive samples from the PBS group falsely showed negative results; (4) 40% of positive samples in the Sputasol group incorrectly show negative results, and negative samples incorrectly show positive results; (5) Only 10% of positive samples from the spithasol boiled group showed a negative result in error. In summary, the PBS group and the Sputasol boiling group showed better effect, and the Sputasol boiling method was slightly superior to the PBS method. Meanwhile, considering that the PBS group treatment steps are complicated and a centrifuge is needed, the Sputasol boiling method is adopted as a rapid pretreatment method for establishing the sputum specimen for ExCad detection.

Example 6 feasibility verification of ExCad-Sp detection with Rapid pretreatment sputum specimens

To determine the sensitivity of the ExCad-Sp assay in clinical diagnosis, we collected a sputum sample from one healthy volunteer to prepare a labeled sample, as follows: to 0.45mL of sputum specimen, 0.05mL of ATCC49619 bacterial suspension (10) ⁷ -10CFU/mL, 10-fold dilution) to produce a solution containing 10 per microliter ³ To 10 ^-3 Labeling of individual streptococcus pneumoniae cells mimics sputum specimens. PBS was used as a negative control instead of bacterial suspension.

To evaluate the specificity of the ExCad-Sp assay in clinical diagnosis, we collected 32 sputum specimens from patients with different respiratory tract infections, including 14 Streptococcus pneumoniae positive and 18 Streptococcus pneumoniae negative sputum specimens. Each sputum specimen was inoculated on a Columbia blood agar plate, a chocolate agar plate and an eosin metablue agar plate, respectively, and incubated at 35℃for 24-72 hours, and then isolated colonies were identified by mass spectrometry (MALDI-TOF MS). The mass spectrometry is used as a diagnostic gold standard.

All of the above specimens were pretreated according to the Sputasol boiling method described in example 5. mu.L of each pre-treated sample was taken for ExCad-Sp analysis. ExCad-Sp detection was first performed on a real-time fluorescent quantitative PCR instrument to capture fluorescent values in real-time. After the reaction is finished, the reaction tube is placed under blue light, the result is read by naked eyes, and a smart phone is used for photographing.

The results were as follows: when using unextracted, labeled mock samples, the limit of detection (LOD) for the ExCad-Sp assay was 1 streptococcus pneumoniae cell per reaction (figures 7,a-d). Since there are 2 copies of the lytA gene in the ATCC49619 genome, LOD corresponds to 2 copies/reaction. Similar results were obtained by visual inspection under blue light (fig. 7 c).

In the specificity verification, only 1 positive specimen (P5) was not clearly distinguished from the negative control by naked eyes under blue light due to the low fluorescence value, and other specimens were all obtained correct detection results except that the specimens were erroneously judged to be negative (fig. 7 e). We compared the consistency of ExCad-Sp method with mass spectrometry using mass spectrometry as gold standard, and obtained an analysis sensitivity of ExCad-Sp of 92.9% (13/14), a specificity of 100% (18/18), a Positive Predictive Value (PPV) of 100% (13/13) and a Negative Predictive Value (NPV) of 94.7% (18/19) (FIG. 7 f).

The main technical advantages of the invention are as follows:

1. rapid pretreatment of sputum specimen

The technical scheme for rapidly preprocessing the sputum specimen is simple to operate, economical and time-saving. The sputum specimen can be liquefied and bacterial nucleic acid in the sputum specimen can be released only by sputum digestion and at 100 ℃ for 10min, and the sputum specimen can be used for nucleic acid detection including fluorescent quantitative PCR and ExCad.

2. Technical advantages of ExCad

The ExCad technology adopts LAMP for pre-amplification and combines with CRISPR/Cas12b mediated detection, and can perform one-step reaction in a single tube at one temperature (52-62 ℃), so that compared with the CRISPR detection by a two-step method, the risk of cross contamination and environmental pollution is greatly reduced, and the turnover time is shortened. In addition, the ExCad technique provides the possibility of detection without nucleic acid extraction relative to the CRISPR-top technique, and a more relaxed primer design approach allows ExCad to be applied to detect any sequence even if the target sequence does not contain the appropriate PAM sites, as the PAM sites can be introduced by our artificially designed loop primers.

3. ExCad-Sp detection method and streptococcus pneumoniae family self-detection kit

Based on the ExCad-Sp detection principle, a streptococcus pneumoniae family self-detection kit is developed. The kit can be used for automatically detecting the nucleic acid of streptococcus pneumoniae in sputum at home, and the whole detection process only needs three steps, namely: (1) rapid pretreatment of sputum specimens (10 min at 100 ℃); (2) Cas12b/gRNA complex preparation (10 min at 37 ℃); and (3) ExCad detection (1 h at 57 ℃). The time from sample acquisition to detection result acquisition by using the kit is less than 1.5 hours. In addition, the self-checking kit does not depend on special instruments and equipment, and only needs a simple temperature control device (for example, water is added into a pot to be boiled to be 100 ℃, the body temperature can be 37 ℃, and a thermos cup matched with a thermometer can be 57 ℃) and a portable LED blue light lamp.

For ExCad, theoretically all disposable consumables and reagents can be supplied with the kit. For example, the oligonucleotides may be stably transported and stored by lyophilization and rehydrated by addition of buffer for use. Furthermore, as mentioned above, the isothermal conditions required for the reaction are easily obtained at home. And the equipment has low cost: 1 bottle of sputum digest is sold at about 40 yuan, which can support the detection of 100 samples (assuming 1mL of each sputum sample), namely 0.4 yuan for each sample; the cost of each isothermal amplification reaction system is about 30 yuan, and one reusable LED blue light lamp is about 10 yuan. In addition, as mass production expands, costs can be further reduced. Therefore, the ExCad-Sp detection method is not only suitable for self-detection at home by individuals, but also suitable for large-scale community monitoring and point-of-care detection.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. The LAMP primer group for detecting streptococcus pneumoniae is characterized by comprising primers F3, B3, FIP, BIP, LF and LB, and the nucleotide sequences of the primers are shown in SEQ ID NO. 1-6 respectively.

2. A nucleic acid molecule for detecting streptococcus pneumoniae, characterized in that it comprises the LAMP primer set, gRNA and ssDNA probes of claim 1;

wherein, the gRNA targets the DNA fragment of streptococcus pneumoniae lytA gene, and the DNA fragment is obtained by adopting the LAMP primer group for amplification;

the ssDNA probe is single-stranded DNA conforming to the principle of trans-cleavage of Cas12b protein.

3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence of the gRNA is set forth in SEQ ID No. 7.

4. The nucleic acid molecule of claim 3, wherein the ssDNA probe has a nucleotide sequence as set forth in SEQ ID No. 8, and wherein the ssDNA probe is labeled with a fluorescent group and a quenching group at the 5 'and 3' ends, respectively; or alternatively, the process may be performed,

the 5 'end and the 3' end of the ssDNA probe are respectively marked with a fluorescent group and biotin.

5. A kit for detecting streptococcus pneumoniae, characterized in that the kit comprises the nucleic acid molecule of any one of claims 2-4 and a Cas12b protein;

preferably, the kit further comprises at least one of LAMP reaction buffer, bst 2.0DNA polymerase, cas12b reaction buffer, and sputum digestate, spathasol.

6. A method for the detection of streptococcus pneumoniae for non-diagnostic purposes, characterized in that it is carried out using a kit according to claim 5, comprising the steps of:

1) Preparation of Cas12b/gRNA complex: mixing Cas12b protein and gRNA with Cas12b reaction buffer, and incubating at 35-39 ℃ for 5-15min to obtain Cas12b/gRNA complex; preferably, the Cas12b protein is an AapCas12b protein, with the reaction conditions: incubating for 10min at 37 ℃;

2) Mixing the LAMP primer set of claim 1, bst 2.0DNA polymerase, LAMP reaction buffer, cas12b/gRNA complex, ssDNA probe and template, reacting at a constant temperature in the range of 52-62 ℃ for at least 20min; preferably, the reaction conditions are: the reaction was carried out at 57℃for 1h.

7. The method according to claim 6, further comprising the step of pre-treating the sample prior to step 1), in particular as follows: adding 1-1.5 times of sputum digestive fluid Sputasol into a sample to be detected, incubating for 5-15min at 100 ℃, and taking the pretreated sample as a template; preferably, the reaction conditions are: incubate at 100℃for 10min.

8. The method according to claim 6 or 7, wherein in the reaction system, the concentration of the primers F3 and B3 is 0.08 to 0.6. Mu.M, the concentration of the primers FIP and BIP is 0.34 to 2.38. Mu.M, the concentration of the primers LF and LB is 0.17 to 1.19. Mu.M, the concentrations of the primers F3 and B3 are the same, the concentrations of the primers FIP and BIP are the same, and the concentrations of the primers LF and LB are the same; preferably, the concentration of primers F3, B3 is 0.17. Mu.M, the concentration of primers FIP, BIP is 0.68. Mu.M, and the concentration of primers LF and LB is 0.34. Mu.M.

9. The method according to claim 8, wherein the total volume of the reacted system is 25 μl, comprising: 12.5. Mu.L of 2 xLAMP reaction buffer, 1. Mu.L of 8U Bst 2.0 enzyme, 0.34-2.38. Mu.M each of primers FIP and BIP, 0.17-1.19. Mu.M each of primers LF and LB, 3-5. Mu.L of primer F3 and B3 each of 0.08-0.6. Mu. M, cas12B/gRNA complex, 0.2-2.0. Mu.L of 100. Mu.M ssDNA probe, 1-5. Mu.L of template, and 25. Mu.L of deionized water are used for the addition, and the concentrations of primers F3 and B3 are the same, the concentrations of primers FIP and BIP are the same, and the concentrations of primers LF and LB are the same; preferably, the total volume of the system of the reaction is 25 μl, comprising: 12.5. Mu.L of 2 XLAP reaction buffer, 1. Mu.L of 8U Bst 2.0 enzyme, 0.68. Mu.M each of primers FIP and BIP, 0.34. Mu.M each of primers LF and LB, 0.17. Mu. M, cas12B each of primers F3 and B3, 3.5. Mu.L of 100. Mu.M ssDNA probe, 1. Mu.L of template, and 25. Mu.L of deionized water;

the preparation method of the Cas12b/gRNA complex comprises the following steps: 15pmol of Cas12b protein and 2 mu L of 10 mu M gRNA are placed in a Cas12b reaction buffer solution, and incubated for 5-15min at 35-39 ℃ to obtain the compound; preferably, the reaction conditions are incubation for 10min at 37 ℃.

10. A nucleic acid molecule for target gene detection, characterized in that the nucleic acid molecule comprises a LAMP primer group for target gene detection, a gRNA and a ssDNA probe, and the nucleic acid molecule is matched with CRISPR-Cas protein and can be used for target gene detection;

the LAMP primer group is designed according to a target gene, comprises an inner forward primer, an inner reverse primer, an outer forward primer, an outer reverse primer, a forward loop primer and/or a reverse loop primer, and modifies a PAM sequence at the 5' end of the loop primer;

the gRNA targets the DNA fragment of the target gene, and the DNA fragment is obtained by adopting the LAMP primer group for amplification;

the ssDNA probe is single-stranded DNA conforming to the principle of trans-cleavage of Cas proteins.

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