CN114381538A - LAMP primer group and detection kit for detecting nocardia meliloti - Google Patents
LAMP primer group and detection kit for detecting nocardia meliloti Download PDFInfo
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Abstract
The invention provides an LAMP primer group and a detection kit for detecting nocardia meliloti. The invention is provided withpbr1The gene is used as a target detection gene of nocardia gangrensis, an LAMP primer is designed and synthesized, the LAMP amplification technology and the CRISPR-Cas12a detection technology are combined for detecting the nocardia gangrensis, and a PAM site sequence TTTA is inserted into a connecting region of a primer FIP or BIP, so that the method is not limited by the PAM site. The detection method provided by the invention can complete detection only by a simple constant-temperature metal bath or water bath and a portable fluorescence reader or a disposable portable test strip without repeated detectionThe complex precision instrument is particularly suitable for on-site and bedside detection.
Description
Technical Field
The invention relates to the field of nucleic acid detection, in particular to an LAMP primer group and a detection kit for detecting nocardia meliloti.
Background
Nocardia is an aerobic, partially acid-fast, gram-positive rod, considered a opportunistic pathogen common in patients with acute or chronic lung infections or suppurative diseases. Nocardia gangrene bacterium (A), (B)Nocardia farcinica) Is one of the most common pathogens causing nocardiosis lung. General will notIdentification of the species level of nocardia is of great importance in clinical diagnosis and treatment, since different species of nocardia have different drug resistance profiles. Furthermore, nocardia meliorati has some phenotypic similarities to gordonia, rhodococcus and fast growing mycobacteria, which can lead to misjudgment of the infectious pathogen and, in turn, misdiagnosis. For many nocardiosis patients, misdiagnosis can lead to a delay in the course of the disease or to improper treatment, resulting in high mortality. The culture of Nocardia gangrene usually requires 2-7 days, which brings difficulties to culture-based identification methods (such as MALDI-TOF MS and the like), and therefore, a simple, rapid and accurate diagnosis method which does not depend on the culture is urgently needed to be established.
In 2004, June M.Brown et al developed a PCR method using specific DNA fragments of 314 bp in length as detection targets to identify Nocardia meliloti clinical isolates. In 2007, Taichi et al established a methodnfa29510 Gene and part of non-open reading frame gene are used as PCR method for detecting target to identify Nocardia meliloti. However, the interpretation of the results of these methods depends on agarose gel electrophoresis, the presence of non-specific amplified bands affects the interpretation of the results, and both PCR amplification and agarose gel electrophoresis require special instruments and are therefore very limited in on-site rapid detection applications.
Loop-mediated isothermal amplification (LAMP) is a specific, efficient and rapid method developed by Notomi et al in 2000, and can complete amplification of a target sequence within 1 hour at a single temperature. The LAMP primer is generally composed of 6 primers designed aiming at 8 target sequence regions, and comprises 2 inner primers FIP and BIP, 2 outer primers F3 and B3, and 2 loop primers LF and LB. LAMP offers more possibilities for rapid diagnosis in the field. However, detection with LAMP amplification alone may give false positive results due to cross contamination, non-specific amplification or primer dimer formation.
In recent years, regularly clustered short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas) systems open the door for new biotechnological applications such as gene editing and transcriptional regulation. Meanwhile, the discovery of the Cas protein with trans-cleavage activity also provides great help for the development of a new generation of nucleic acid detection platform. For example, the Cas 12-based DETECTR detection platform and HOLMES one hour low cost multi-purpose high efficiency system have been used for the detection of Human Papillomavirus (HPV) and single nucleotide polymorphisms. Similarly, the slerlock detection platform based on Cas13 and the DETECTR based on Cas14 were also developed for the detection of bacteria, viruses and fungi. These CRISPR-based detection methods have higher specificity than specific sequence amplification methods alone (such as PCR, LAMP, etc.) due to increased recognition of target sequences by CRISPR RNA (crRNA), and can be detected even in the presence of single-base mutations.
Disclosure of Invention
The invention aims to provide an LAMP primer group and a detection kit for detecting nocardia meliloti.
The invention has the following conception: to be provided withpbr1The gene is used as a target detection gene of nocardia meliloti, an LAMP primer is designed and synthesized, and the LAMP amplification technology and the CRISPR-Cas12a detection technology are combined for the detection of nocardia meliloti.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a LAMP primer set for detecting nocardia meliloti, the LAMP primer set comprising:
outer forward primer F3: 5'-GCGGTGAGCAGTGACGT-3', respectively;
outer reverse primer B3: 5'-ACCCGGCACCAGGAGT-3', respectively;
inner forward primer FIP: 5'-CCATGTCGTAGGCGACCAGCTCGCCACCATGCGGAAAC-3', respectively;
inner reverse primer BIP: 5'-AGTGTGCGCGACGAACAACCGGCGCCATGGTGGGGTT-3', respectively;
loop primer LF: 5'-GAGACCGCGTTGTCCCC-3', respectively;
the loop primer LB: 5'-ACGCTGACGATGGCACT-3' are provided.
In a second aspect, the invention provides a detection reagent or kit containing the LAMP primer set.
In a third aspect, the invention provides a nocardia meliloti nucleic acid detection kit based on CRISPR-Cas12a technology, which comprises the LAMP primer group, crRNA, Cas12a protein, a single-stranded DNA probe double-labeled by a fluorescent group and a fluorescence quenching group; introducing a PAM locus into the FIP or BIP of the LAMP primer group; alternatively, the first and second electrodes may be,
the kit comprises the LAMP primer group, crRNA, Cas12a protein, a fluorescent group and a biotin double-labeled single-stranded DNA probe; and a PAM site is introduced into the FIP or BIP primer of the LAMP primer group.
Preferably, the sequence of the crRNA is: 5'-UAAUUUCUACUAAGUGUAGAUUCGCCACCAUGCGGAAACGGCU-3' are provided.
Preferably, the sequence of the single-stranded DNA probe is: 5'-TTTATTTATTT-3' are provided.
More preferably, the 5 'end of the single-stranded DNA probe is modified with a 6-FAM or FITC group, and the 3' end is modified with BHQ 1.
More preferably, the 5 'end of the single-stranded DNA probe is modified with 6-FAM or FITC group, and the 3' end is modified with biotin.
In one embodiment of the present invention, the nucleotide sequence of the primer FIP introduced into the PAM site is: 5'-CCATGTCGTAGGCGACCAGCTCGCCACCATGCGGAAAC-3' are provided.
In a fourth aspect, the invention provides a nocardia meliloti nucleic acid detection method (including non-disease diagnosis purpose) based on CRISPR-Cas12a technology, which comprises the following steps:
1) extracting DNA in a sample to be detected;
2) performing LAMP amplification reaction by using the DNA extracted in the step 1) as a template and using an LAMP primer group in the kit;
3) mixing the amplification product obtained in the step 2) with crRNA, Cas12a protein and a single-stranded DNA probe doubly labeled by a fluorescent group and a fluorescence quenching group, and carrying out fluorescence intensity detection after reacting for a period of time.
In a fifth aspect, the invention provides a nocardia meliloti nucleic acid detection method (including non-disease diagnosis purpose) based on CRISPR-Cas12a and lateral flow chromatography technology, comprising the following steps:
1) extracting DNA in a sample to be detected;
2) performing LAMP amplification reaction by using the DNA extracted in the step 1) as a template and using an LAMP primer group in the kit;
3) mixing the amplification product obtained in the step 2) with a reaction buffer solution, a complex formed by crRNA and Cas12a protein and a single-stranded DNA probe doubly labeled by a fluorescent group and a fluorescence quenching group, and carrying out fluorescence intensity detection after reacting for a period of time.
In the invention, the lateral flow chromatography detection test strip consists of a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad.
Wherein, the binding pad is coated with streptavidin modified nano-gold particles;
the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with biotinylated bovine serum albumin, and the quality control line is coated with an anti-carboxyfluorescein (FAM) antibody or an anti-Fluorescein Isothiocyanate (FITC) antibody.
In the present invention, the reaction system of the LAMP amplification reaction comprises, in 25. mu.L: 12.5. mu.L of 2 × isothermal amplification buffer, 1. mu.LBst2.0 (8000U/mL), 0.4. mu.M primers F3 and B3, 0.8. mu.M primers LF and LB, 1.6. mu.M primers FIP and BIP, 1. mu.L DNA template, supplemented to 25. mu.L with DNase and RNase free Deionized Water (DW).
Preferably, the LAMP amplification reaction conditions are: the reaction was carried out at 70 ℃ for 40 minutes.
The step 3) comprises the following steps: the amplification product was mixed with reaction buffer, a complex formed by crRNA and Cas12a protein, and a fluorophore and biotin double-labeled single-stranded DNA probe, and reacted at 37 ℃ for 8 minutes.
The 100 μ L CRISPR/Cas12a assay system includes: mu.L of 2 XNEBuffer 2.1, 2.5. mu.L of 10. mu.M fluorophore-labeled single-stranded DNA probe (ssDNA) (fluorescence read: 5 '-end modified 6-FAM or FITC/3' -end modified BHQ 1; or lateral flow biosensor read: 5 '-end modified 6-FAM or FITC/3' -end modified biotin), 18. mu.L of CRISPR-Cas12a/crRNA complex, 27.5. mu.L of deionized water, 2. mu.L of LAMP product or deionized water (blank). The reaction was carried out at 37 ℃ for 8 minutes.
In a sixth aspect, the present invention providespbr1The gene is used as a detection target gene of nocardia meliloti.
Wherein the content of the first and second substances,pbr1ginseng on NCBI for gene encoded proteinReferred to by sequence number WP _ 011210687.1.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the detection method provided by the invention can be completed only by a simple constant-temperature metal bath or water bath and a portable fluorescence reader or a disposable portable test strip, does not need a complex precise instrument, and is suitable for field and bedside detection. In particular, the lateral flow biosensor is more convenient to carry than a fluorescence reader, and is more suitable for on-site and bedside detection.
And (II) inserting a PAM site sequence TTTA into a connecting region of the primer FIP or BIP, so that the method is not limited by the PAM site, namely, the method can detect any nucleotide sequence, and the crRNA is correspondingly designed according to the insertion position of the PAM site whether the sequence contains a proper PAM site or not.
Drawings
FIG. 1 is a schematic diagram of the work flow and reaction principle of CRISPR-CLA analysis in the preferred embodiment of the present invention.
FIG. 2 is a schematic diagram of the LFB readout CRISPR-CLA reaction result in the preferred embodiment of the present invention. Wherein (A) the structure of LFB. (B) The principle of LFB for CRISPR-CLA outcome visualization. (C) Practical application of LFB.
FIG. 3 is a graph showing the effectiveness of the CRISPR-CLA assay of Nocardia gangreniformis in a preferred embodiment of the present invention. Wherein (A) confirmation of LAMP amplification. (B) And (3) performing fluorescence detection on the Nocardia gangrene CRISPR-CLA. (C) And detecting results of the Nocardia gangrene CRISPR-CLA lateral flow biosensor.
FIG. 4 is the best crRNA screening for the CRISPR-CLA assay of Nocardia Farcinica in the preferred embodiment of the present invention.
FIG. 5 is the best ssDNA probe and working concentration screen for the detection of Nocardia Farcinica CRISPR-CLA in the preferred embodiment of the present invention. Wherein, nt is nucleotide.
FIG. 6A shows Nocardia Farcinica in a preferred embodiment of the inventionpbr1Optimal reaction temperature screening for LAMP Pre-amplification of genes (59 ℃ and 60 ℃).
FIG. 6B shows Nocardia Farcinica in a preferred embodiment of the inventionpbr1Optimal reverse for LAMP pre-amplification of genesTemperature screening (61 ℃ C. and 62 ℃ C.) was performed.
FIG. 6C shows Nocardia Farcinica in a preferred embodiment of the inventionpbr1Optimal reaction temperature screening for LAMP Pre-amplification of genes (63 ℃ and 64 ℃).
FIG. 6D shows Nocardia Farcinica in a preferred embodiment of the inventionpbr1Optimal reaction temperature screening for LAMP Pre-amplification of genes (65 ℃ and 66 ℃).
FIG. 6E shows Nocardia Farcinica in a preferred embodiment of the inventionpbr1Optimal reaction temperature screening for LAMP Pre-amplification of genes (67 ℃ C. and 68 ℃ C.).
FIG. 6F shows Nocardia Farcinica in a preferred embodiment of the inventionpbr1Optimal reaction temperature screening for LAMP preamplification of genes (69 ℃ and 70 ℃).
FIG. 7 shows the sensitivity of the CRISPR-CLA assay of Nocardia gangreniformis in the preferred embodiment of the present invention. Wherein, (A) sensitivity of fluorescence method Nocardia gangrene CRISPR-CLA detection. (B) Sensitivity of lateral flow Nocardia gangreniformis CRISPR-CLA detection.
FIG. 8 is a graph showing the feasibility of the CRISPR-CLA reaction of Nocardia gangreniformis in clinical specimen examination according to the preferred embodiment of the present invention. Wherein, (A) the fluorescence method Nocardia gangrene CRISPR-CLA detection result. (B) And (3) detecting the result of the CRISPR-CLA of the nocardia meliloti by a lateral flow method. S1-S20, Nocardia gangrene simulates a positive sample; s21, a patient sample infected by nocardia meliloti; N1-N20, Nocardia Farcinica negative sample.
FIG. 9 shows the results of 41 clinical samples tested by PCR in the preferred embodiment of the present invention. Wherein, the positive sample has a 314 bp band on the agarose gel. Horizontal lines are used to aid in interpretation of results. S1-S20, Nocardia gangrene simulation positive specimen; s21, a sample of a patient with nocardiosis; N1-N20, Nocardia Farcinica negative sample. M, DL2000 DNA molecular marker; p, positive control; n, negative control.
FIG. 10 shows the result of the LAMP amplification primer optimization experiment in the preferred embodiment of the present invention.
Detailed Description
The invention uses CRISPR-CLA platform to diagnose nocardiosis caused by nocardia meliloti infection quickly and accurately, and the method is named as melioidosisNocardia CRISPR-CLA detection method. The invention reports Nocardia gangrene aiming at the first timepbr16 LAMP primers are designed in 8 regions of the gene. To meet the requirements of CRISPR-CLA detection, a PAM site TTTA was added to the ligation region of the FIP primer (fig. 1 and table 1).
In order to verify the effectiveness of the LAMP primer, LAMP amplification is carried out by adopting a nocardia fargesii reference strain IFM 10152. The negative control used an equal volume of DW instead of template DNA. The amplification effect was monitored using a real-time turbidimeter (Loopamp, LA-320 c). Our results show that the designed primer can amplify the target sequence within 40 min (FIG. 3A). Then, CRISPR detection is performed with LAMP products. And setting a blank control to verify the effectiveness of the CRISPR reaction system. As a result, the fluorescence signal of the positive reaction was found to be significantly increased within 8 minutes, whereas the fluorescence signals of the negative control and the blank control were not significantly increased (FIG. 3B).
Visualization of nocardia meliloti CRISPR-CLA results was performed on LFB. First, CRISPR reaction systems using LAMP positive and negative products were incubated with a metal bath at 37 ℃ for 8 minutes. The results were then read on LFB according to the CRISPR-CLA workflow. As shown in FIG. 3C, red bands appear on both the quality control line and the detection line of the positive reaction, and only red bands appear on the quality control line of the negative reaction. The whole process of the nocardia meliloti CRISPR-CLA detection can be completed within 70 minutes.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.
Reagents and instruments used in the following examples:
wizard genomic DNA purification kits were purchased from Promega, USA. The 10 XNEBuffer 2.1 and EnGen Lba Cas12a enzymes were purchased from New England Biolabs, USA. The DNA isothermal amplification kit was purchased from Vietnam Biotech (Tianjin) Ltd. The Nanodrop ND-1000 microspectrophotometer and the thermostatted metal bath were purchased from Beijing Saimei technologies, Inc. In the present invention, a real-time fluorescent quantitative PCR instrument (QuantStaudio 6 Flex) was used as a fluorescence reader.
Example 1 Nocardia Farcinica LAMP amplimer and crRNA design
Discovery by bioinformatic analysispbr1The gene (the coded product is PQQ-binding-like beta-propeller-repeat protein, GenBank accession number: WP-011210687.1) has high conservation and specificity in Nocardia Farcinica, so the gene is selected as the target gene for detection.
One set of targets was designed online with the aid of Primer Explorer software (5 th edition) (http:// Primer Explorer. jp/e /)pbr1LAMP primers for genes. Primers were tested for specificity using BLASTn software. Oligo Analyzer software (version 3.1) (http:// eu. idtdna. com/calc/Analyzer) was used to ensure that the primers did not form secondary structures and primer dimers that affected the detection results. Then, 3 different lengths of CRISPR RNA (crRNA) were designed according to the LAMP primer sites. LAMP primers were synthesized and purified by Biotechnology Limited, crRNA was synthesized and purified by Tianyihui Biotechnology Limited, and both primers and crRNA were purified by HPLC grade. The sequences of the primers and crRNA are shown in Table 1.
TABLE 1 primer, crRNA and ssDNA Probe sequences
aThe NF-FIP primer modifies a PAM locus TTTA in a connecting region;bnt, nucleotide.
Example 2 CRISPR-CLA assay
The design and reaction principle of CRISPR-CLA are shown in figure 1. In the CRISPR-CLA system, the PAM site TTTA of Cas12a is modified at the linker region of the FIP primer. The FIP primer for CRISPR-CLA detection consists of three regions, including the F1c region (5 'end, complementary to the F1 region), the inserted PAM site (linker region, independent of the target sequence) and the F2 region (3' end). Thus, any target sequence can be detected using the CRISPR-CLA method of the invention, even if these sequences do not contain a suitable PAM site. After LAMP pre-amplification, the amplicon contains a PAM site obtained from the modified FIP primer, and can be used for guiding the Cas12a/crRNA compound to recognize and combine with a target sequence. If the target sequence is recognized and crRNA binds to it, Cas12a is activated and the trans-cleavage activity is activated, resulting in cleavage of the single-stranded DNA reporter. The fluorescent signal is then released and captured by a real-time fluorescence reader. Another approach is to read CRISPR-CLA results with a lateral flow biosensor within 2 minutes (fig. 1 and 2). Thus, the entire CRISPR-CLA assay procedure can be completed in 70 minutes, including rapid DNA extraction (20 minutes), LAMP pre-amplification (40 minutes), CRISPR-based detection (8 minutes) and result reading (fluorescence, instant; lateral flow, 2 minutes).
The CRISPR-CLA assay comprises two steps: first, the target sequence is exponentially amplified using the LAMP technique. The 25 μ L LAMP reaction system contained: 12.5. mu.L of 2 × isothermal amplification Buffer (BF), 1. mu.LBst2.0 (8000U/mL), 0.4. mu.M primers F3 and B3, 0.8. mu.M primers LF and LB, 1.6. mu.M primers FIP and BIP, 1. mu.L DNA template, the total reaction volume was made up to 25. mu.L with DNase and RNase free Deionized Water (DW). In the negative control reaction, the template DNA was replaced with an equal volume of DW. The CRISPR-Cas12a/crRNA complex (CRISPR-Cas 12a/crRNA complex is prepared by 250. mu.L of 2 XNEBuffer 2.1, 245. mu.L of DW, 0.375. mu.L of 100. mu.M Cas12a and 5. mu.L of 10. mu.M crRNA, mixed and incubated in a thermostatic metal bath or water bath at 37 ℃ for 10 minutes) is stored at 4 ℃ for no more than 18 hours before use. The configuration of the CRISPR/Cas12a based detection system was then performed. The 100 μ L CRISPR/Cas12a assay system includes: mu.L of 2 XNEBuffer 2.1, 2.5. mu.L of 10. mu.M fluorophore-labeled single-stranded DNA (ssDNA) reporter (fluorescence read: 5 '-end modified 6-FAM or FITC/3' -end modified BHQ 1; or lateral flow biosensor read: 5 '-end modified 6-FAM or FITC/3' -end modified biotin), 18. mu.L of CRISPR-Cas12a/crRNA complex, 27.5. mu.L of deionized water, 2. mu.L of LAMP product or deionized water (blank). The reaction was carried out at 37 ℃ for 8 minutes.
Fig. 1 is a schematic diagram of the work flow and reaction principle of CRISPR-CLA analysis. (A) CRISPR-CLA workflow. The whole reaction process of the CRISPR-CLA analysis can be within 70 minutesAnd (4) finishing. (B) Primer and crRNA design for Nocardia gangreniformis CRISPR-CLA analysis. By Nocardia gangrene IFM 10152pbr1The gene is used as a target, and LAMP primers are designed. The figure is given onlypbr1Partial nucleotide sequence of gene (904 to 1182 bp). The right and left arrows represent the sense and antisense strands, respectively. Inserted PAM sites (TTTA) are highlighted on a light gray background. crRNA is boxed. (C) Introduction of PAM sites into amplicons by LAMP preamplification. Dashed arrows point to the corresponding amplification products. (D) Principle of CRISPR-CLA analysis. Firstly, a PAM locus is introduced into a target amplicon by modifying an FIP primer, and the target amplicon is exponentially increased by LAMP pre-amplification. In the second step, the Cas12a/crRNA complex is stably formed. In a third step, the target amplicon is recognized and bound to Cas12a/crRNA complex to activate Cas12a, and the activated Cas12a cleaves single-stranded dna (ssdna) reporter.
Example 3 CRISPR-CLA lateral flow biosensor reading
Visualization of CRISPR-CLA results on LFB: as a more convenient approach, the results of CRISPR-CLA can also be shown on LFB. The principle of the readout of the results on the LFB is shown in fig. 2. And (3) putting the CRISPR cleavage reaction solution into a constant-temperature metal bath or water bath at 37 ℃ for incubation for 8 minutes, then dropwise adding 8 mu L of reaction product onto a sample pad of LFB, and then dropwise adding 2-3 drops of running buffer solution. The running buffer was moved by capillary action along the nitrocellulose membrane and rehydrated the immobilized GNPs-SA on the conjugate pad. In the negative reaction, ssDNA is not cleaved and all FAM components are captured by anti-FAM on the control line, whereas biotin binds to GNPs-SA. Thus, only the control line shows a red band in the negative result. In a positive reaction, activated Cas12a cleaves ssDNA and FAM and biotin are separated. Biotin-BSA adsorbs the isolated biotin/GNPs-SA complexes, while anti-FAM adsorbs the isolated FAM groups and uncleaved ssDNA. In positive results, red bands appeared on both control and test lines.
A portable Lateral Flow Biosensor (LFB) enables convenient and intuitive reading of the results of CRISPR-CLA (fig. 2). LFB reference documents (Zhu, X.; Wang, X.; Li, S.; Luo, W.; Zhang, X.; Wang, C.; Chen, Q.; Yu, S.; Tai, J.; Wan, Wan.)g, Y., Rapid, Ultrasensitive, and Highly Specific Diagnosis of COVID-19 by CRISPR-Based Detection. ACS sensors 2021, 6(3) 881-888). The LFB consists of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad (fig. 2A). Streptavidin-modified gold nanoparticles (GNPs-SA) were placed on the conjugate pad as indicator reagents. An anti-carboxyfluorescein antibody (anti-FAM) and biotinylated bovine serum albumin (Biotin-BSA) were distributed on a nitrocellulose membrane as a detection line (T line) and a quality control line (C line), respectively.
First, 8 μ L of CRISPR-CLA reaction product was added to a sample pad of LFB. Then, 2 to 3 drops (about 50. mu.L) of a running buffer (the running buffer is composed of 100 mM PBS, 1% Tween-20, pH 7.4) were dropped onto the sample pad, and the reaction solution was allowed to flow from bottom to top along the nitrocellulose membrane. The CRISPR-CLA detection results can be read visually within 2 minutes. Positive results showed red bands on both line C and line T, and negative results showed only red bands on line C (fig. 2C).
The principle of LFB for CRISPR-CLA result visualization is shown in fig. 2B.
Example 4 specificity and sensitivity of the Nocardia Farcinica CRISPR-CLA reaction
82 Nocardia gangrene strains and 50 Nocardia non-gangrene strains were collected by the biosafety laboratory of the center for disease prevention and control in China for infectious disease prevention and control, and used to test the specificity of the Nocardia gangrene CRISPR-CLA assay (Table 2).
Genomic DNA of Nocardia Farcinica reference strain IFM 10152 was extracted and double-stranded DNA (dsDNA) concentration was quantified using Nanodrop ND-1000. Sensitivity assays for Nocardia gangreniformis CRISPR-CLA detection were performed by serially diluting dsDNA from 1 ng/. mu.L to 1 fg/. mu.L with 10-fold concentrations of Deionized Water (DW). Each reaction used 1. mu.L of dsDNA as template. The detection Limit (LOD) of the CRISPR-CLA of nocardia meliloti is tested by using real-time fluorescent CRISPR-CLA and lateral-flow CRISPR-CLA detection methods, respectively.
All strains used in the present invention were stored in 20% (w/v) glycerol broth and frozen at-70 ℃ before DNA extraction. DNA was extracted using Wizard genomic DNA purification kit according to the instructions.
TABLE 2 information on the strains used according to the invention
a Performing sequence comparison by blastn software after Sanger sequencing to identify the strains;bp, positive and N, negative.
Specificity of Nocardia gangrene CRISPR-CLA detection:
132 isolates, including bacteria and fungi, were tested for Nocardia gangreniformis CRISPR-CLA (Table 2). The Nocardia gangrene IFM 10152 is used as a positive control, and a negative control and a blank control are set. The results show that all positive results are from Nocardia gangrene, and the Nocardia non-melissa is negative. The result shows that the Nocardia gangrene CRISPR-CLA detection method has higher specificity and has no cross reaction on Nocardia gangrene bacteria. It is worth noting that the result of the lateral flow Nocardia gangreniformis CRISPR-CLA detection is completely consistent with that of the fluorescence method.
Sensitivity of nocardia melitensis CRISPR-CLA detection:
the genomic DNA of Nocardia gangrene reference strain IFM 10152 is diluted in a multiple ratio (1 ng-1 fg, 10 times dilution) and used for determining the detection lower limit of the Nocardia gangrene CRISPR-CLA detection method. Both fluorescence and lateral flow CRISPR-CLA were tested with similar results (figure 7).
Example 5 detection of clinical samples Using the CRISPR-CLA reaction of nocardia meliloti
In order to evaluate the feasibility of the Nocardia gangrene CRISPR-CLA detection method in clinical specimen detection, 41 sputum specimens are subjected to CRISPR-CLA detection, and the CRISPR-CLA detection method comprises 20 simulated Nocardia gangrene positive specimens (S1-S20), 1 Nocardia disease patient specimen (S21) and 20 Nocardia gangrene negative specimens (N1-N20). Detection for determining CRISPR-CLA of nocardia melilotiEffectiveness of the method on clinical specimens. Mixing 100 μ L of 105The bacterial suspension of the CFU/ml strain IFM 10152 is mixed with 900 mu L of nocardia farinosa negative sputum to form a simulated nocardia farinosa positive sputum specimen.
The sputum specimen was liquefied in a 4% sodium hydroxide solution, and then subjected to DNA extraction using a DNA purification kit. Positive, negative and blank controls were set for each experiment. The same DNA template extracted from clinical specimens was used for the analysis of Nocardia gangreniformis CRISPR-CLA and the reported detection of PCR (Brown, J. M.; Pham, K. N.; McNeil, M.; Lasker, B. A., Rapid identification of Nocardiac cosmetic isolates by a PCR assay targeting a 314-base-pair spectra-specific DNA fragment).Journal of clinical microbiology 2004, 42 (8), 3655-60）。
The results of the CRISPR-CLA assay of Nocardia gangrene are shown in FIG. 8, with S1-S21 producing a positive result and N1-N20 producing a negative result. The results of the fluorescence method and the lateral flow method were consistent. The result shows that the Nocardia gangrene CRISPR-CLA detection method is a method suitable for detecting Nocardia gangrene in clinical samples.
The reported PCR detection results are shown in FIG. 9, in which 42.8% (9/21) of Nocardia gangrene positive samples were false negative, and 5.0% (1/20) of Nocardia gangrene negative samples were false positive. The appearance of some non-specific bands due to cross-amplification confounded the interpretation of the results. The above experimental results show that the effect of the CRISPR-CLA method is obviously superior to that of the PCR method when detecting Nocardia gangrene in clinical samples (Table 3).
TABLE 3 comparison of the results of the detection of Nocardia Farcinica in clinical samples by the use of the method for detecting Nocardia Farcinica CRISPR-CLA and the PCR method
Example 6 screening and optimization of optimal reaction conditions for Nocardia Farcinica CRISPR-CLA
1. Screening for optimal crRNA
3 crRNAs of different lengths were designed based on the amplification site (Table 1), and then the detection efficiency of the different crRNAs was compared. Negative and blank controls were set for each crRNA test reaction. As shown in FIG. 4, the fluorescence value of crRNA3 was the highest after 8 minutes of reaction, but the fluorescence signal of crRNA1 was not significantly enhanced compared to the three crRNAs. This suggests that crRNA3 (43 nt in length) was most efficiently detected.
2. Optimal ssDNA Probe and working concentration screening
Based on the principle of Cas12a trans-cleavage, 3 ssDNA probes of different lengths were designed (table 1). The detection effect of 3 probes at 2 working concentrations was tested. As shown in FIG. 5, the fluorescence intensity increased with increasing probe length and working concentration. The results show that the fluorescence of the 11 nt probe is highest at 250 nM concentration.
3. Optimal reaction temperature for LAMP Pre-amplification
To determine the optimal reaction temperature for LAMP pre-amplification, Nocardia Farcinica IFM 10152(100 pg genomic DNA per reaction) pairs were usedpbr1Isothermal amplification is performed. The amplification efficiency was tested at 59-70 deg.C (1 deg.C differential) and the amplification product was monitored using a real-time turbidimeter. As shown in FIGS. 6A to 6F, the amplification rate was the fastest at 70 ℃ and the product content was high, so that 70 ℃ was regarded aspbr1Optimal temperature for LAMP pre-amplification.
Example 7 optimization of LAMP amplification primers
According to Nocardia gangrene reference strain IFM 10152pbr1Gene sequence 2 sets of LAMP amplification primers were designed (Table 4). To screen a more preferable primer set, Nocardia gangrensis IFM 10152 genomic DNA (100 pg/10 pg genomic DNA per reaction) and DW (negative control) were used as amplification templates, reacted at 65 ℃ for 1 hour, and the amplification products were monitored by a real-time turbidimeter. As shown in the figure, although the reaction system containing primer set 1 significantly amplified the positive control at 31 minutes (100 pg genomic DNA) and 34 minutes (10 pg genomic DNA) of the reaction, the negative control also amplified at 44 minutes and 54 minutes, indicating that primer set 1 may cross-react with the primers to cause a false positive result, so primer set 1 should be excluded. The reaction system containing the primer set 2 was allowed to proceed for 37 minutes (100 pg genomic DNA) and 42 minutes (10 pg genomic DNA) in the reactionDNA), the positive control was significantly amplified and the negative control was not amplified within 1 hour of the reaction, indicating that primer set 2 was more specific than primer set 1, so primer set 2 was selected (fig. 10).
TABLE 4 optimization of LAMP primers
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Reference documents:
1、Qiu, X.;Xu, S.;Liu, X.;Han, L.;Zhao, B.; Che, Y.; Han, L.; Hou, X.; Li, D.; Yue, Y.; Chen, S.; Kang, Y.; Sun, L.; Li, Z., A CRISPR-based nucleic acid detection platform (CRISPR-CPA): Application for detection of Nocardia farcinica. Journal of applied microbiology 2021.
2、Li, S.; Huang, J.; Ren, L.; Jiang, W.; Wang, M.; Zhuang, L.; Zheng, Q.; Yang, R.; Zeng, Y.; Luu, L. D. W.; Wang, Y.; Tai, J., A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): Application for the diagnosis of COVID-19. Talanta 2021, 233, 122591.
3、Zhu, X.; Wang, X.; Li, S.; Luo, W.; Zhang, X.; Wang, C.; Chen, Q.; Yu, S.; Tai, J.; Wang, Y., Rapid, Ultrasensitive, and Highly Specific Diagnosis of COVID-19 by CRISPR-Based Detection. ACS sensors 2021, 6 (3), 881-888.
4、Brown, J. M.;Pham, K. N.;McNeil, M. M.; Lasker, B. A., Rapid identification of Nocardia farcinica clinical isolates by a PCR assay targeting a 314-base-pair species-specific DNA fragment. Journal of clinical microbiology 2004, 42 (8), 3655-60.
sequence listing
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Claims (10)
1. An LAMP primer group for detecting nocardia meliloti, which is characterized in that the LAMP primer group comprises:
outer forward primer F3: 5'-GCGGTGAGCAGTGACGT-3', respectively;
outer reverse primer B3: 5'-ACCCGGCACCAGGAGT-3', respectively;
inner forward primer FIP: 5'-CCATGTCGTAGGCGACCAGCTCGCCACCATGCGGAAAC-3', respectively;
inner reverse primer BIP: 5'-AGTGTGCGCGACGAACAACCGGCGCCATGGTGGGGTT-3', respectively;
loop primer LF: 5'-GAGACCGCGTTGTCCCC-3', respectively;
the loop primer LB: 5'-ACGCTGACGATGGCACT-3' are provided.
2. The kit for detecting the nocardia meliloti nucleic acid based on CRISPR-Cas12a technology, which is characterized by comprising the LAMP primer group, crRNA, Cas12a protein, a fluorescent group and a fluorescence quenching group double-labeled single-stranded DNA probe as claimed in claim 1; introducing a PAM locus into the FIP or BIP of the LAMP primer group; alternatively, the first and second electrodes may be,
the kit comprises the LAMP primer group of claim 1, crRNA, Cas12a protein, a fluorophore and a biotin double-labeled single-stranded DNA probe; introducing a PAM locus into the FIP or BIP of the LAMP primer group;
wherein, the sequence of the crRNA is as follows: 5'-UAAUUUCUACUAAGUGUAGAUUCGCCACCAUGCGGAAACGGCU-3', respectively;
the sequence of the single-stranded DNA probe is as follows: 5'-TTTATTTATTT-3' are provided.
3. The kit according to claim 2, wherein the nucleotide sequence of the primer FIP introducing the PAM site is: 5'-CCATGTCGTAGGCGACCAGCTCGCCACCATGCGGAAAC-3' are provided.
4. The kit according to claim 2 or 3, wherein the 5 'end of the single-stranded DNA probe is modified with 6-FAM or FITC group, and the 3' end is modified with BHQ 1; alternatively, the first and second electrodes may be,
the 5 'end of the single-stranded DNA probe is modified with 6-FAM or FITC group, and the 3' end is modified with biotin.
5. The nocardia meliloti nucleic acid detection method based on CRISPR-Cas12a technology is characterized in that the method is used for non-disease diagnosis and comprises the following steps:
1) extracting DNA in a sample to be detected;
2) performing LAMP amplification reaction by using the DNA extracted in the step 1) as a template and using the LAMP primer group in the kit according to any one of claims 2 to 4;
3) mixing the amplification product obtained in the step 2) with a reaction buffer solution, a complex formed by crRNA and Cas12a protein and a single-stranded DNA probe doubly labeled by a fluorescent group and a fluorescence quenching group, and carrying out fluorescence intensity detection after reacting for a period of time.
6. A method for detecting nocardia meliloti nucleic acid based on CRISPR-Cas12a and lateral flow chromatography, which is for non-disease diagnosis purposes, characterized by comprising the following steps:
1) extracting DNA in a sample to be detected;
2) performing LAMP amplification reaction by using the DNA extracted in the step 1) as a template and using the LAMP primer group in the kit according to any one of claims 2 to 4;
3) mixing the amplification product obtained in the step 2) with a reaction buffer solution, a compound formed by crRNA and Cas12a protein, a fluorescent group and a biotin double-labeled single-stranded DNA probe, reacting for a period of time, adding the reaction product onto a lateral flow chromatography detection test strip, dropwise adding 2-3 drops of running buffer solution, enabling the reaction product to flow along the test strip under the action of capillary, and observing a color development result within 2 minutes.
7. The method of claim 6, wherein the lateral flow assay test strip is comprised of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad;
wherein, the binding pad is coated with streptavidin modified nano-gold particles;
the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with biotinylation bovine serum albumin, and the quality control line is coated with an anti-carboxyfluorescein antibody or an anti-isothiocyanic fluorescein antibody.
8. The method according to any one of claims 5 to 7, wherein the reaction system of the LAMP amplification reaction of step 2) comprises, in terms of 25. mu.L: 12.5. mu.L of 2 × isothermal amplification buffer, 1. mu.L of 8000U/mL Bst2.0, 0.4. mu.M primers F3 and B3, 0.8. mu.M primers LF and LB, 1.6. mu.M primers FIP and BIP, 1. mu.L DNA template, supplemented to 25. mu.L with deionized water without DNase and RNase;
LAMP amplification reaction conditions: the reaction was carried out at 70 ℃ for 40 minutes.
9. The method according to any one of claims 5 to 7, wherein step 3) comprises: the amplification product was mixed with reaction buffer, a complex formed by crRNA and Cas12a protein, and a fluorophore and biotin double-labeled single-stranded DNA probe, and reacted at 37 ℃ for 8 minutes.
10.pbr1The use of the gene as a detection target gene of nocardia meliloti;
wherein the content of the first and second substances,pbr1the reference sequence of the protein encoded by the gene at NCBI is numbered WP _ 011210687.1.
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| CN115746112A (en) * | 2022-07-14 | 2023-03-07 | 山东第一医科大学附属省立医院(山东省立医院) | Nocardia specific antigen protein, serological diagnosis kit and application thereof |
| CN116574841A (en) * | 2023-06-27 | 2023-08-11 | 广西大学 | Method for detecting multiple pathogenic bacteria of CRISPR/Cas12a cutting system of non-specific CrRNA |
| CN117305512A (en) * | 2023-11-30 | 2023-12-29 | 中国农业大学 | Nucleic acid detection method for detecting soybean plant MON87705 |
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| CN113388691A (en) * | 2021-07-23 | 2021-09-14 | 中国疾病预防控制中心传染病预防控制所 | Nucleic acid detection method based on PCR amplification and CRISPR-Cas12a and application |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115746112A (en) * | 2022-07-14 | 2023-03-07 | 山东第一医科大学附属省立医院(山东省立医院) | Nocardia specific antigen protein, serological diagnosis kit and application thereof |
| CN115746112B (en) * | 2022-07-14 | 2023-10-13 | 山东第一医科大学附属省立医院(山东省立医院) | Nocardia specific antigen protein, serological diagnosis kit and application thereof |
| CN116574841A (en) * | 2023-06-27 | 2023-08-11 | 广西大学 | Method for detecting multiple pathogenic bacteria of CRISPR/Cas12a cutting system of non-specific CrRNA |
| CN116574841B (en) * | 2023-06-27 | 2023-12-15 | 广西大学 | Method for detecting multiple pathogenic bacteria of CRISPR/Cas12a cutting system of non-specific CrRNA |
| CN117305512A (en) * | 2023-11-30 | 2023-12-29 | 中国农业大学 | Nucleic acid detection method for detecting soybean plant MON87705 |
| CN117305512B (en) * | 2023-11-30 | 2024-03-05 | 中国农业大学 | Nucleic acid detection method for detecting soybean plant MON87705 |
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