CN114134218B - Fluorescent detection method based on CRISPR-Cas12a - Google Patents
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Abstract
The invention discloses a CRISPR-Cas12 a-based fluorescence detection method and application, wherein PCR amplification and CRISPR-Cas12a fluorescence detection are combined to detect Giardia gaisoniana, a target DNA fragment is amplified through PCR, and the specific fluorescence detection of CRISPR-Cas12a is realized by using the trans-cutting activity of Cas12a protein. The detection platform can be used for identifying the Nocardia gainstarch and has the characteristics of high detection speed, high sensitivity and high specificity.
Description
Technical Field
The invention discloses a method for detecting pathogen nucleic acid based on combination of CRISPR-Cas12a and PCR, belonging to the field of molecular biology and microbiology.
Background
Nocardia is an opportunistic pathogen that can cause severe infections of almost all organs, including traumatic infections, lung disease, and brain abscesses. In recent years, nocardia infection has increased significantly in immunocompromised patients, including aids late stage, diabetic patients and organ transplant patients. Nocardia californica (Nocardia cyriaciigioeorgica) is increasingly a common Nocardia infection pathogen. Because the sequences of Nocardia species are highly similar, the Nocardia is difficult to identify to a species level by traditional separation culture and biochemical tests, and different species of Nocardia have different antibacterial drug resistance modes, so that a rapid and specific detection method for identifying the Nocardia bacterium Gelsense and guiding early-stage clinical reasonable medication is urgently needed.
The CRISPR-Cas system composed of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas protein) is an acquired immune system in bacteria or archaea. The CRISPR-Cas system is useful not only for gene editing but also for nucleic acid detection of pathogens. The Cas12a protein has a trans-cleavage activity, the Cas12a protein is specifically combined with a targeting double-stranded DNA (dsDNA) under the guidance of crRNA and activates the trans-cleavage activity, non-specific single-stranded DNA (ssDNA) is indiscriminately cleaved, and a fluorescent signal is emitted after a ssDNA probe is cleaved by modifying a fluorescent group and a quenching group at two ends of the ssDNA, so that the CRISPR-Cas12a system can be used as a tool for detecting nucleic acid. The DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) is a nucleic acid detection method established based on CRISPR-Cas12a, and specifically identifies Human Papillomavirus (HPV) 16 and 18. The CRISPR-Cas12a fluorescence detection method can be used for detecting different pathogens by designing different crRNA sequences to guide Cas12a protein to be combined with different targeting dsDNA sequences.
Based on the problems in the prior art, the invention aims to establish a nucleic acid detection method combining polymerase chain amplification reaction (PCR) and CRISPR-Cas12a detection and apply the nucleic acid detection method to the detection of Nocardia.
Disclosure of Invention
In view of the above objects, the present invention firstly provides a method for detecting nucleic acid based on non-diagnostic purpose using CRISPR-Cas12a technology, the method comprising the steps of:
(1) Performing polymerase chain amplification reaction (PCR) by using a coding gene of a protein of which protein _ id is VFA97995.1 in a genome of the Gaersoniensis as a target gene;
(2) Reacting a polymerase chain amplification product obtained in the step (1), a Cas12a protein, a fluorescent group and a fluorescence quenching group double-labeled single-stranded DNA probe and a crRNA molecule in a CRISPR-Cas12a technology detection system, wherein the sequence of the crRNA molecule is shown as SEQ ID NO. 3;
(3) And (3) detecting the fluorescence intensity of the reaction system in the step (2).
The method combines PCR amplification and CRISPR-Cas12a fluorescence detection, is named as 'CRISPR-PCR', the primer and the probe are designed based on protein _ id: VFA97995.1 encoding gene of the Nocardia gainstangsonii, the whole genome access of the Nocardia gainstangsonii: LR21597.3, and the encoding gene of the protein _ id: VFA97995.1 is positioned between 1870620-169493 bp, and is named as Ncc1 gene by the applicant. The method provided by the invention can be used for detecting the Nocardia gainstanjia. Firstly, amplifying a target DNA fragment according to a PCR primer; specific crRNA is then designed according to the conserved sequence to guide binding of Cas12a protein to targeted dsDNA to achieve detection of genococcus dahliae.
In a preferred embodiment, the sequence of the upstream primer and the sequence of the downstream primer used in the PCR are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The PCR amplification interval is 79bp-266bp of the Ncc1 gene, and the length of the target fragment amplified by PCR is 187bp.
In a more preferred embodiment, the sequence of the single-stranded DNA probe in step (2) is shown in SEQ ID NO.4, and the single-stranded DNA probe is labeled with a fluorescent group 6-FAM at the 5 'end and a fluorescence quenching group BHQ1 at the 3' end.
In another preferred embodiment, the annealing temperature for the polymerase chain amplification reaction of step (1) is 60-65 ℃.
More preferably, the annealing temperature of the polymerase chain amplification reaction in step (1) is 65 ℃.
In a further preferred embodiment, the detection of fluorescence intensity in step (3) is a real-time fluorescence quantitative detection method.
Secondly, the present invention provides a combination of nucleic acid molecules for use in the above method, said combination of nucleic acid molecules comprising: the PCR primer sequence includes upstream PCR primer shown in SEQ ID No.1, downstream PCR primer shown in SEQ ID No.2 and crRNA molecule shown in SEQ ID No. 3.
Finally, the invention provides a nucleic acid detection kit containing the nucleic acid molecule combination, and the kit further comprises: cas12a protein, a fluorescent group and a fluorescence quenching group double-labeled single-stranded DNA probe.
In a preferred embodiment, the single-stranded DNA probe is labeled with a fluorescent group 6-FAM at the 5 'end and a fluorescence quencher BHQ1 at the 3' end.
In a more preferred embodiment, the DNA probe has the sequence shown in SEQ ID NO. 4.
The method for detecting the Gaerson kichenoca bacteria based on CRISPR-PCR fluorescence has extremely high sensitivity, and the lower detection limit is 10 -3 ng. The PCR-PCR fluorescence detection is positive when the CRISPR-PCR fluorescence detection is carried out on 60 Gaerssen Kinokia sp; when the method is applied to the detection of clinical samples, the concentration of the clinical samples can be detected to be 10 6 -10 4 CFU/mL Nocardia Galersonii with a detection limit of 10 4 CFU/mL; the concentration of the blood plate separation culture can be detected to be 10 6 -10 5 CFU/mL of Nocardia murinus gaelsen. Therefore, compared with separation culture, the fluorescence detection method of the CRIPSR-PCR of the Nocardia californica has better sensitivity, the detection time is greatly shortened, and the detection can be completed within two hours. The method provided by the invention is simple, convenient, rapid and efficient, is easy to be widely developed on site and at the base level, and has excellent application prospect.
Drawings
Fig. 1 is a schematic diagram of the working flow and principle of a CRISPR-PCR fluorescence detection system. The CRIPSR-PCR fluorescence detection process comprises three steps, namely genome extraction, PCR amplification of a target fragment and CRISPR fluorescence detection. The whole detection process can be completed within two hours. Principle of CRISPR-PCR fluorescence detection: firstly, amplifying a target DNA fragment by PCR; and then the Cas12a protein is combined with crRNA to form a CRISPR-Cas12a/crRNA complex, the Cas12a protein is combined with the targeting double-stranded DNA under the guidance of the crRNA to activate the trans-cleavage activity of the targeting double-stranded DNA, a single-stranded DNA probe is indiscriminately cleaved, the probe emits fluorescence after cleavage, and a real-time fluorescence quantitative PCR (polymerase chain reaction) instrument can be used for capturing a fluorescence signal.
FIG. 2 primer and crRNA position and sequence for CRISPR-PCR fluorescence detection of Nocardia gairsonii.
FIG. 3 optimal annealing temperature Screen for PCR amplification. The annealing temperature is screened by temperature gradient PCR, the temperature range is 60-65 ℃, and the strip with the annealing temperature of 65 ℃ is bright and almost has no non-specific amplification.
FIG. 4 sensitivity of CRISPR-PCR fluorescence detection of Nocardia gairsonii. Ten-fold serial dilution of DNA template of Galson Xinka in the concentration of 10ng, 1ng and 10ng successively -1 ng、10 -2 ng、10 -3 ng、10 -4 ng, NC is a negative control without DNA template.
FIG. 5 shows the specificity of CRISPR-PCR fluorescence detection of Nocardia gelsense. 1-60 represent 60 strains of Nocardia gairsoni (including 7 standard strains and 53 clinical strains). The ordinate represents the amount of change in fluorescence value in a real-time fluorescent quantitative PCR apparatus at 37 ℃ for 20 minutes of reaction; NC is negative control.
FIG. 6.25 strains of Nocardia non-Gelsenzhikh; the positive control is the standard strain DSM 44484 of Nocardia gainstanz; NC is negative control.
FIG. 7.19 strain of non-Nocardia bacteria, positive control Nocardia gainstanxinella standard strain DSM 44484; NC is negative control.
Fig. 8 clinical application of crispr-PCR fluorescence detection. The final concentration of the Nocardia bacterium Gelsemii in the clinical specimen is 10 in turn 6 CFU/mL-10 2 CFU/mL; NC is negative control. The ordinate represents the change in fluorescence value after 20 minutes of reaction at 37 ℃.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of protection defined by the claims of the present invention.
The invention discloses a CRISPR-PCR working process and a detection principle:
as shown in fig. 1, the CRISPR-PCR detection method comprises the following three steps: 1. extracting a sample genome for 15 minutes; PCR amplification, 70 min; 3. fluorescence detection based on CRIPSR, 20 min. The whole CRISPR-PCR fluorescence detection process can be completed within 2 hours.
After genome DNA is extracted, a target gene fragment is amplified by PCR so as to improve the detection sensitivity; in the CRISPR detection stage, cas12a protein and crRNA are firstly combined at 37 ℃ to form a CRISPR-Cas12a/crRNA complex, cas12a is specifically combined with a target sequence in a target fragment under the guidance of the crRNA to activate the trans-cleavage activity of the Cas12a protein, a non-specific ssDNA probe is indiscriminately cleaved, ssDNA (5 '-6-FAM-TTTTTT-BHQ 1-3') with a sequence modified with a fluorescent group and a quenching group as shown in SEQ ID NO.4 is cleaved to emit fluorescence, and a real-time fluorescence quantitative PCR instrument is used for detecting a fluorescence signal.
Reagents and apparatus used in the invention:
DNA extraction kit
Genomic DNA Purification Kit was purchased from Promega, USA. PCR reaction mixture 2 × Premix Ex Taq (Probe qPCR) and DL 2000DNA Marker were purchased from Bao bioengineering (Beijing) Ltd. CRISPR reaction buffer 10 XNEBuffer 2.1 and Cas12a protein
Lba Cas12a (Cpf 1) was purchased from New England Biolabs, USA.
The real-time fluorescence quantitative PCR instrument QuantStudio 6Flex, the PCR instrument Veriti 96 and the spectrophotometer NanoDrop-1000 are products of Sammerfo USA. The electrophoresis apparatus is a product of Beijing Junyi Oriental electrophoresis apparatus Co. The Gel imaging System Bio-Rad Gel Dox XR is a product of Bio-Rad corporation, USA. The constant temperature metal bath is a product of Beijing gold, silver and apricot Life sciences GmbH.
The strains used in the experiments of the invention:
the strains used in the experiment of the invention are collected and preserved in a biological safety laboratory of infectious disease prevention and control institute of China disease prevention and control center, and the details are shown in Table 1.
TABLE 1 strains used in the experiments of the invention
Example 1 construction of fluorescent detection platform for CRISPR-Cas12a
1. Sequence design
PCR primers and crRNA sequences are designed according to the genome of the standard strain DSM 44484 of Nocardia gainstanji, the amplified target gene is named as Ncc1 by the applicant, the protein _ id encoded by the gene is VFA97995.1 Nocardia whole genome access of Nocardia gainstanji, LR21597.3, the Ncc1 gene is positioned between 1870620-1871693bp, the polymerase chain amplification interval is 79bp-266bp of the Ncc1 gene, and the length of the amplified target fragment is 187bp. The specificity of the PCR primers was verified by Primer-BLAST in NCBI. The sequences and positions of the PCR primers and the crRNA design are shown in Table 2 and FIG. 2.
TABLE 2 Galson-Kyocardia primers and crRNA sequences
Note: NN represents degenerate bases.
PCR amplification
The total reaction system for PCR amplification was 20. Mu.L, containing 10. Mu.L of Premix Ex Taq (Probe qPCR), 7. Mu.L of enzyme-free water, 1. Mu.L of each of the upstream and downstream primers, and 1. Mu.L of each of the DNA templates. Amplification was performed using a PCR instrument Veriti 96 programmed for 95 ℃ pre-denaturation for 5min,35 cycles of 95 ℃ denaturation for 30s, 65 ℃ annealing for 30s,72 ℃ extension for 30s, and 72 ℃ final extension for 7min. The PCR amplification products were verified by electrophoresis on 2% agarose gel.
CRISPR fluorescence detection reaction system
CRISPR-Cas12a/crRNA complexes comprising 100nM crRNA and 75nM Cas12a protein were first configured and incubated in a metal bath at 37 ℃ for 10 min. The total reaction system for CRISPR fluorescence detection comprises 50 μ L of 2 XNEBuffer 2.1, 18 μ L of CRISPR-Cas12a/crRNA complex, 2.5 μ L of ssDNA probe with the concentration of 10 μ M, 27.5 μ L of enzyme-free water and 2 μ L of PCR amplification product.
Results of CRISPR detection
And (3) performing CRISPR fluorescence detection by using a real-time fluorescence quantitative PCR instrument. And (3) reacting the CRISPR reaction system for 20 minutes in a real-time fluorescent quantitative PCR instrument at 37 ℃, and reading a fluorescent signal. If the signal is positive, the fluorescence signal changes; if negative, there is no change in the fluorescence signal.
5. Optimization of CRISPR-PCR amplification reaction conditions of Gaersheng Nocardia
And screening the optimal annealing temperature of the PCR amplification product. The optimal annealing temperature is selected by adopting gradient PCR amplification, and the temperature range is between 60 and 65 ℃. After PCR amplification, the PCR amplification product was detected by 2% agarose gel electrophoresis. As a result, as shown in FIG. 3, the band of interest was bright and almost non-specifically amplified under the reaction condition of 65 ℃.
Example 2 sensitivity of the CRISPR-PCR fluorescence detection System of Nocardia mureinhei
Detection of 10-fold serial dilutions of genomic DNA of the standard strain of Nocardia gelsenensis DSM 44484 at a concentration ranging from 10ng to 10ng by CRISPR-PCR -4 ng. As a result, as shown in FIG. 4, the limit of detection of the CRISPR-PCR fluorescence detection of Nocardia gaiersonii was 10 -3 ng。
Example 3 specificity of CRISPR-PCR fluorescence detection System of Nocardia guilliermondii Gelsense
The specificity of the CRISPR-PCR fluorescence detection method of the Gelsemiq nocardia was evaluated by using 60 Gelsemiq nocardia (including 7 standard strains and 53 clinical isolates), 25 other strains of the Nocardia and 19 DNA of other genera as templates. If the change of the fluorescence value is positive; if there is no change in the fluorescence value, it is negative.
As shown in FIGS. 5 to 7, the fluorescence values of 60 Nocardia gaiersonii strains in CRISPR-PCR fluorescence detection were positive (see FIG. 5); the other 25 Nocardia strains and 19 other bacteria strains have no change of fluorescence signals when CRISPR-PCR fluorescence detection is carried out, and are negative (see FIGS. 6-7). The result shows that the CRISPR-PCR fluorescence detection system has good specificity.
Example 4 application of Nocardia Galersonii CRISPR-PCR fluorescent detection System to detection of clinical specimens
In order to evaluate the sensitivity of the CRISPR-PCR fluorescence detection method for detecting clinical samples, the collected negative sputum samples are equally divided into 5 parts, each part is 900 mu L, 100 mu L of Ten-fold serial diluted bacterial suspension of Nocardia Gelsemii is added, and the final concentration of the bacterial suspension is 10 6 CFU/mL to 10 2 CFU/mL. Blood plate separation culture was also used as a control.
As shown in FIG. 8, the CRISPR-PCR detected the concentration of 10 in the clinical specimen 6 -10 4 CFU/mL of Nocardia gaelsen with a detection limit of 10 4 CFU/mL; the concentration detected by blood plate separation culture is 10 6 -10 5 CFU/mL of Nocardia gaelsen. Therefore, compared with separation culture, the sensitivity of the CRIPSR-PCR fluorescence detection method of the Kyokhstan gesii is better, the detection time is greatly shortened, and the detection can be completed within two hours.
Sequence listing
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<120> fluorescent detection method based on CRISPR-Cas12a and application thereof
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Claims (10)
1. A method for detecting nucleic acids using CRISPR-Cas12a technology for non-diagnostic purposes, comprising the steps of:
(1) Carrying out polymerase chain amplification reaction by taking a coding gene of a protein of which protein _ id is VFA97995.1 in the genome of the Gaersin Xinnocardia as a target gene;
(2) Reacting the polymerase chain amplification product obtained in the step (1), a single-stranded DNA probe doubly labeled by a Cas12a protein, a fluorescent group and a fluorescence quenching group and a crRNA molecule in a CRISPR-Cas12a technical detection system, wherein the sequence of the crRNA molecule is shown as SEQ ID NO. 3;
(3) And (3) detecting the fluorescence intensity of the reaction system in the step (2).
2. The method according to claim 1, wherein the upstream primer used in the pcr reaction has a sequence shown in SEQ ID No.1, and the downstream primer has a sequence shown in SEQ ID No. 2.
3. The method according to claim 2, wherein the sequence of the single-stranded DNA probe in step (2) is shown in SEQ ID NO.4, and the single-stranded DNA probe is labeled with a fluorophore 6-FAM at the 5 'end and a fluorescence quencher BHQ1 at the 3' end.
4. The method of any one of claims 1 to 3, wherein the annealing temperature of the polymerase chain amplification reaction of step (1) is 60 to 65 ℃.
5. The method of claim 4, wherein the annealing temperature of the PCR of step (1) is 65 ℃.
6. The method according to any one of claims 1 to 3, wherein the detection of the fluorescence intensity in step (3) is a real-time quantitative fluorescence detection method.
7. A combination of nucleic acid molecules for use in the method of claim 1, wherein said combination of nucleic acid molecules comprises: the PCR primer sequence includes upstream PCR primer shown in SEQ ID No.1, downstream PCR primer shown in SEQ ID No.2 and crRNA molecule shown in SEQ ID No. 3.
8. A nucleic acid detection kit comprising the combination of nucleic acid molecules of claim 7, wherein said kit further comprises: cas12a protein, a fluorescent group and a fluorescence quenching group double-labeled single-stranded DNA probe.
9. The kit according to claim 8, wherein the single-stranded DNA probe is labeled with a fluorophore 6-FAM at the 5 'end and a fluorescence quencher BHQ1 at the 3' end.
10. The kit of claim 9, wherein the DNA probe has a sequence as shown in SEQ ID No. 4.
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