CN117965687A - Amplification method of single-stranded DNA product and application thereof - Google Patents
Amplification method of single-stranded DNA product and application thereof Download PDFInfo
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Abstract
The invention discloses an amplification method of a single-stranded DNA product and application thereof, belonging to the field of biological detection, wherein a section of non-human genome sequence irrelevant to a detection target sequence is screened out from a genome database to be used as a universal single-tailed primer; connecting the 3 'end of the universal single tailing primer sequence to the 5' end of one pathogen nucleic acid sequence primer sequence; the target specific primer is subjected to exponential amplification under the annealing temperature condition; then, carrying out linear amplification by using a universal single-tailed primer, and reacting to generate a single-stranded DNA product; meanwhile, based on the single-stranded DNA amplification method and combining with a CRISPR-Cas system, a pathogen nucleic acid detection method without PAM sequence dependence is established; the invention solves the problems of large design difficulty and low amplification efficiency of primers in single-stranded DNA amplification by the traditional method; meanwhile, the problem that the CRISPR-Cas system depends on the PAM sequence when the target sequence is identified is solved.
Description
Technical Field
The invention relates to the field of biological detection, in particular to pathogen nucleic acid detection, and particularly relates to an amplification method of a single-stranded DNA product and application thereof.
Background
Methods for generating single-stranded DNA (ssDNA) based on PCR techniques, the preparation of ssDNA was initially performed by asymmetric PCR techniques, which mainly include primer concentration asymmetric PCR, conventional thermal asymmetric PCR, and staggered thermal asymmetric PCR. The Linear-After-The-Exponential (Late-PCR) method, i.e. The post-exponential Linear amplification PCR technique, is an advanced optimization form of The asymmetric polymerase chain reaction, is an improvement of The asymmetric PCR technique, and adopts primer pairs with different concentrations to amplify on The basis of The asymmetric PCR technique. The specific process of the Late-PCR technology is as follows: the restriction primer and the excess primer are amplified from the exponential phase, wherein the amplification efficiency is similar to that of conventional PCR; once the restriction primer is exhausted, the reaction is suddenly switched to linear amplification and the single stranded product continues through a number of additional thermal cycles, thereby efficiently producing ssDNA. At present, the PCR technology is widely applied to the biomedical technical fields of pathogen nucleic acid detection, gene chips and the like.
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system is a highly efficient gene editing tool, consisting of CRISPR loci, cas effector proteins and guide RNAs. The discovery of the trans-cleavage property of the CRISPR-Cas system at present promotes the rapid development of a nucleic acid detection technology based on the system, such as a Nobel chemical prize Jennifer Doudna in 2020 and a team thereof establish a DNA endonuclease-targeted CRISPR trans-report detection system DETECTR by utilizing LbCas a, the CRISPR trans-report detection system DETECTR is combined with CrRNA to form a complex, double-stranded DNA which has a PAM sequence and is complementary to CrRNA is cleaved, the trans-cleavage activity of the Cas12a is activated, the Cas12a/CrRNA/DNA target is combined into a ternary complex, the Cas12a plays the activity of single-stranded deoxyribonuclease (ssDNase), and the single-stranded DNA is subjected to nonspecific cleavage, so that HPV types 16 and 18 in a clinical sample can be effectively detected. The rapid and high-sensitivity detection method of the CRISPR system has been realized by utilizing the activity characteristics of Cas proteins and specifically detecting target DNA or RNA target sequences.
Therefore, the technology of identifying the DNA target in the CRISPR-Cas system can be adopted, the characteristic of trans-cleavage activity of Cas12a protein (the identification of the single-stranded DNA target can be free from dependence on PAM sequences) is utilized, the detection of the single-stranded DNA target is carried out by the CRISPR/Cas12a coupled PCR technology, and the trans-activity cleavage can be carried out in a constant temperature reaction, and the fluorescent signal detection is completed by a fluorescent probe in a cleavage system.
However, there are problems with single strand amplification by the LATE-PCR technique, in which the LATE-PCR primer design follows certain design principles such as: the restriction primer annealing temperature Tm L, the excess primer annealing temperature Tm X and the amplicon annealing temperature Tm A meet the following relation to have higher amplification efficiency Tm L-TmX≥5℃、23℃≥TmA-TmX not less than 13 ℃, and for pathogen specific sequences, the primer design is difficult to meet the above conditions.
Therefore, there is a need to improve the above PCR amplification techniques for single stranded DNA generation to achieve a method that efficiently, conveniently, simply generates single stranded targets, and efficiently couples CRISPR/Cas systems for pathogen nucleic acid detection verification.
Disclosure of Invention
The invention aims to solve the problem of single-stranded DNA generation by improving the LATE-PCR technology, namely difficult design of primers for different templates, and simultaneously can achieve single-stranded target amplification efficiency as high as that of the LATE-PCR technology, provides a novel method for single-stranded DNA amplification, and provides a single-stranded DNA detection method based on a CRISPR/Cas system aiming at the characteristic that CRISPR/Cas12a recognizes single-stranded and does not need PAM sequences.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, a method for amplifying a single stranded DNA product, the method comprising:
Step 1, designing a universal single-tailed primer: screening a segment of non-human genome sequence irrelevant to a detection target sequence in a genome database, and searching a segment of segment from the sequence to serve as a universal single-tailed primer; the sequence of the universal single-tailed primer is as follows: 5'-TTTTTAGTTTACATTGTGC-3';
Step 2, designing a primer pair according to a pathogen nucleic acid sequence; the design principle of common PCR primers is met, and the Tm value between the upstream primer and the downstream primer of the pathogen nucleic acid sequence primer meets the delta Tm of less than or equal to 5 ℃;
Step 3, designing target specific primers: connecting the 3 'end of the universal single tailing primer sequence to the 5' end of one pathogen nucleic acid sequence primer sequence;
Step 4, PCR amplification: in the first stage, target specific primers are amplified exponentially under higher annealing temperature conditions; in the second stage, after the exponential amplification is finished, the annealing temperature is reduced, linear amplification is carried out by using a universal single-tailed primer, and finally, a single-stranded DNA product is generated through reaction;
wherein, the Tm value of the universal single-tailed primer is lower than that of the pathogen nucleic acid sequence primer, and the difference between the Tm value and the Tm value is more than or equal to 5 ℃.
Preferably, the sequence length of the universal single tailing primer is 18-25bp.
The PCR amplification system in the step 4 is as follows: 5U/. Mu.L Taq enzyme, 0.3. Mu.L; 2.5mM each dNTP,1 μl; target specific primer, 0.3 μl×2; 2 mu L of universal single-tailed primer; 10 XPCR Buffer, 2. Mu.L; ddH 2 O, 12.1. Mu.L; 2. Mu.L of DNA template;
PCR amplification procedure: the first stage: 95 ℃ for 3min; cycling for 30 times at 95 ℃,10s, 65 ℃ and 30 s; and a second stage: 72 ℃,20s; cycling for 20-30 times at 95 ℃,10s, 55 ℃,15s, 72 ℃ and 20s; 72℃for 4min.
The invention designs a single-tailed primer aiming at the nucleic acid sequence of each pathogen target, wherein the single-tailed primer is a segment of non-human genome sequence and has poor homology with the pathogen target sequence, the sequence is simply called a universal single-tailed primer, the universal single-tailed primer is only added on one primer of the target, the design principle of the primers is simpler than LATE-PCR, besides the design principle of the common PCR primer, the design points that the Tm value of the single-tailed primer is lower than that of the pathogen nucleic acid sequence primer, the GC content at the 3 'end of the single-tailed primer is higher (namely 3-5G/C bases exist at the 3' end) and the like are required to be met, firstly, the target specific primer carries out an exponential first amplification stage, and then carries out a linear second amplification stage with the universal single-tailed primer to realize the efficient amplification of single-stranded DNA,
In a second aspect, there is provided the use of a method for amplification of single stranded DNA products in the detection of pathogen nucleic acids, in particular: and (3) generating a single-stranded DNA product by using the single-stranded DNA product amplification method, combining the single-stranded DNA product amplification method with a CRISPR/Cas system, namely combining the single-stranded DNA product with Cas12a and Cr-RNA to form a complex, activating trans-cleavage activity, and cutting fluorescence to generate a signal to finish fluorescence signal detection.
Based on the single-stranded DNA amplification method and combining with a CRISPR-Cas system, a pathogen nucleic acid detection method without PAM sequence dependence is established; the detection method is suitable for a CRISPR/Cas system which has PAM sequence requirements on double-stranded DNA sequence identification and does not need PAM sequence for single-stranded DNA sequence identification.
The invention has the following technical effects: (1) The invention adds a universal single-tailed primer at the tail end of one target primer to realize the exponential amplification based on the target specific primer pair to generate a double-stranded DNA product with a universal sequence, and then uses the universal primer to perform linear amplification to generate a single-stranded DNA product; solves the problems of the traditional method that the design difficulty of primers is high and the amplification efficiency is low in the single-stranded DNA amplification.
(2) The invention establishes a pathogen nucleic acid detection method without PAM sequence dependence based on the single-stranded DNA amplification method and combining with a CRISPR-Cas system. Solves the problem of dependence on PAM sequence when the CRISPR-Cas system recognizes target sequence, and promotes the application of the CRISPR-Cas system in pathogen nucleic acid detection. The method provided by the invention provides a novel and convenient nucleic acid sequence amplification and detection method concept for the technical field of molecular diagnosis.
Drawings
FIG. 1 is a schematic general flow diagram of the present invention;
FIG. 2 is a schematic diagram of a method for generating single-stranded DNA detection based on universal single-tailed asymmetric amplification according to the present invention;
FIG. 3 is a graph showing the detection of fluorescent signals for the verification of a novel coronavirus target by the method established by the present invention;
FIG. 4 is a graph showing the detection result of fluorescent signals verified on a syncytial virus target by the method established by the invention;
FIG. 5 is a graph showing the detection result of fluorescent signals for the verification of a alphavirus target by the method established by the invention;
FIG. 6 is a graph showing the detection result of fluorescent signal for the verification of the B-stream virus target by the method established by the invention;
FIG. 7 is a fluorescent signal detection result of the method established by the invention for verifying the secondary influenza 2 target;
FIG. 8 is a fluorescent signal detection result of the method established by the invention for verifying the secondary influenza 3 target;
FIG. 9 is a fluorescent signal detection result of metapneumovirus target verification by the established method of the present invention;
FIG. 10 shows the results of the application of the method of the present invention in the detection of syncytial virus samples;
FIG. 11 is a graph showing the results of an application of the method of the present invention in the detection of an B-stream virus sample;
FIG. 12 shows the results of the application of the method of the invention in the detection of metapneumovirus samples.
Detailed Description
The following detailed description and drawings of the present invention will be presented in terms of detailed embodiments and procedures based on the technical scheme of the present invention, but the scope of the present invention is not limited to the following specific examples, and the terms used in the present invention are only for the purpose of describing the specific examples and are not intended to limit the present invention.
FIG. 1 is a schematic diagram of the overall flow of the invention, and FIG. 2 is a schematic diagram of the method for detecting single-stranded DNA based on the general single-tailed asymmetric amplification of the invention; a method for amplifying a single-stranded DNA product comprises the following steps:
1. Design of single tailing primer:
(1) Screening a segment of non-human genome sequence irrelevant to a detection target sequence in a genome database, and searching a segment of segment from the sequence as a universal single tailing primer (below 25 bp), namely a tailing primer serving as a target primer;
(2) Designing primers according to nucleic acid sequences of various pathogens, wherein the difference of Tm values between upstream and downstream primers is small (delta Tm is less than or equal to 5 ℃), namely, the design principle of common PCR primers is satisfied;
(3) Introducing a universal single-tailed primer: uniformly connecting the 3 'end of the universal single tailing primer sequence to the 5' end of the downstream primer sequence of the target sequence, and adhering to the primer design principle: the Tm value of the universal single-tailed primer is lower than that of the pathogen nucleic acid sequence primer, the difference of the Tm values is more than or equal to 5 ℃, and the GC content at the 3' end of the universal single-tailed primer is higher, so that the initiation reaction can be facilitated; the design is carried out while avoiding primer dimer, self stem loop structure and the like.
After the primer design is finished, the amplification condition of the primer is initially verified by a PCR technology and a verification technology of a PAGE gel electrophoresis method.
2. Method for single-stranded amplification establishment and verification
Similar to the LATE-PCR principle, pathogen templates are first amplified exponentially by double-stranded amplification primers (target specific primer pairs), single-stranded templates are amplified, and single-stranded amplification is achieved by single-stranded templates. During amplification verification, the whole single-stranded amplification experimental condition needs to be optimized first: for example, the ratio between the addition amount of the target specific primer and the addition amount of the universal single-tailed primer, the whole annealing temperature in the whole PCR process and the like are optimized, and the single-strand amplification condition is observed through a verification technology of PAGE gel electrophoresis.
3. Application method establishment of CRISPR/Cas coupled by single-stranded amplification method
The nucleic acid detection method based on CRISPR system is being developed more widely, wherein Cas12a recognizes single strand without PAM sequence, enzyme activity of non-specifically cleaving single strand DNA (ssDNA) is activated, and any ssDNA nearby can be cleaved indiscriminately.
After single-stranded amplification, the single-stranded DNA product is mixed with a Cas system and a fluorescent probe system, namely, the single-stranded DNA product is combined with a corresponding target CrRNA, and is combined with Cas12a protein to form a Cas12a-CrRNA ternary complex, trans-cleavage activity is activated, fluorescence is cut to generate signals, the fluorescent signal curve rates measured in different reaction times are counted and compared through a fluorescence detection method, and the phenomenon of the result is observed under the irradiation of a gel imager or an ultraviolet lamp immediately after the reaction is finished, so that the result visualization is realized.
Example 1: verification of single-stranded DNA-new crown pathogen target by utilizing universal single-tailed asymmetric amplification technology
The generation of single-stranded DNA is realized by using a general single-tailed asymmetric amplification method, and in the embodiment, a DNA plasmid is artificially synthesized by adopting a novel coronavirus, so that the feasibility of the nucleic acid detection method is verified.
The reaction system:
TABLE 1PCR System design
* Primer final concentration: 10. Mu.M;
TABLE 2 Cas-FQ System design
Q-PCR conditions: 37-30 s; 37-30 s [ 60cycles ]
PCR reaction conditions:
Table 3 PCR temperature program
Universal single tailed primer (SEQ ID No. 1): 5'-TTTTTAGTTTACATTGTGC-3' (Tm: 54.4 ℃ C.)
Target specific primer sequences (new coronaviruses):
XG-F(SEQ ID No.2)：5’-GGTGAACGTGTACGCCAAGCT-3’(Tm：68.8℃)
XG-R (SEQ ID No. 3): 5'-TTTTTAGTTTACATTGTGCCCACTACCTGGCGTGGTTTGT-3' (Tm: 68.6 ℃ C.; total Tm:74.9 ℃ C.)
The reaction process comprises the following steps:
Firstly, determining the dosage of a new crown pathogen target primer and a universal single-tailed primer, then preparing a PCR system, wherein the dosage is shown in a table 1, the PCR temperature degree adopts a two-step circulation method, and in the first stage, the target specific primer is subjected to exponential amplification under the condition of higher annealing temperature; in the second stage, after the exponential amplification is finished, the annealing temperature is reduced, and the linear amplification is carried out by using a universal single-tailed primer; the number of copies of the DNA template in the PCR system was 10 3 copies/. Mu.L, and the specific PCR conditions are shown in Table 3.
After the PCR amplification is completed, single-stranded DNA products are generated, and further verification is performed by adopting a fluorescence detection method. The Cas system and the fluorescent probe FQ system were subjected to a premix operation and then added with a single-stranded DNA product, as shown in table 2, i.e., crRNA of the DNA product bound to the corresponding target, and Cas12a was coupled to form a ternary complex to cleave single-stranded DNA, and fluorescence signal detection was completed by qPCR reaction conditions at 37 ℃ for 1 hour, sharing a positive group to which the single-stranded DNA product was added and a blank group to which water was substituted for the single-stranded DNA product as a result for comparison. The results are shown in fig. 3, the strong fluorescent signal appears in fig. 3, clearly distinguished from the blank, and the results are observed under irradiation of ultraviolet lamps, and the feasibility and the good specificity of the method of the invention are verified for the target.
In the fluorescent signal detection method of this embodiment, the fluorescent signal is collected by a 7300Plus real-time fluorescent quantitative PCR instrument.
Example 2 validation of Single-stranded DNA-syncytial Virus target production Using Universal Single-tailed asymmetric amplification technology
The feasibility of the method was examined for a syncytial virus target according to the reaction system, reaction conditions, etc. in example 1. The results are shown in FIG. 4, the fluorescent signal appears in FIG. 4, and is clearly distinguished from the blank, and the results are observed under the irradiation of ultraviolet lamps, which indicates that the detection of the nucleic acid target is feasible and has good specificity.
The copy number of the DNA template in the PCR system is 10 3 copies/. Mu.L; in the fluorescent signal detection method of this embodiment, the fluorescent signal is collected by a 7300Plus real-time fluorescent quantitative PCR instrument.
Target specific primer sequences (syncytiviruses):
HB-S1-F(SEQ ID No.4)：5’-CCAGCAAATACACTATTCAACGT-3’(Tm：63.2℃)
HB-S1-R (SEQ ID No. 5); 5'-TTTTTAGTTTACATTGTGCGTCTTCCCTTCCTAACCTGGAC-3' (Tm: 65.5 ℃ C.; total Tm:73.2 ℃ C.)
Example 3 validation of Single-stranded DNA-A-stream Virus target Using Universal Single-tailed asymmetric amplification technique
The feasibility of the method was examined with the alphavirus target according to the reaction system, reaction conditions, etc. in example 1. The results are shown in FIG. 5, the fluorescent signal appears in FIG. 5, and the fluorescent signal is clearly distinguished from the blank control, and the result is observed under the irradiation of an ultraviolet lamp, which shows that the detection of the nucleic acid target is feasible and has good specificity.
The copy number of the DNA template in the PCR system is 10 3 copies/. Mu.L; in the fluorescent signal detection method of this embodiment, the fluorescent signal is collected by a 7300Plus real-time fluorescent quantitative PCR instrument.
Target specific primer sequence (a stream):
JL-S1-F(SEQ ID No.6)：5’-TGAACAGATTGCCGACTCCCAG-3’(Tm：68℃)
JL-S1-R (SEQ ID No. 7): 5'-TTTTTAGTTTACATTGTGCTGTTCACTCGATCCAGCCATTT-3' (Tm: 66.3 ℃ C.; total Tm:73.1 ℃ C.)
Example 4 validation of Single-stranded DNA-Equipped Virus target Using Universal Single-tailed asymmetric amplification technique
The feasibility of the method was examined with respect to the B-stream virus target according to the reaction system, reaction conditions, etc. in example 1. The results are shown in FIG. 6, the fluorescent signal appears in FIG. 6, and is clearly distinguished from the blank, and the results are observed under the irradiation of ultraviolet lamps, which indicates that the detection of the nucleic acid target is feasible and has good specificity.
The copy number of the DNA template in the PCR system is 10 3 copies/. Mu.L; in the fluorescent signal detection method of this embodiment, the fluorescent signal is collected by a 7300Plus real-time fluorescent quantitative PCR instrument.
Target specific primer sequence (b stream):
YL-S1-F(SEQ ID No.8)：5’-CCTGCTTGCTCGTAGTATGGTC-3’(Tm：66℃)
YL-S1-R (SEQ ID No. 9): 5'-TTTTTAGTTTACATTGTGCCCAACCATAGAGTACTCCTCAAC-3' (Tm: 63.6 ℃ C.; total Tm:72.2 ℃ C.)
Example 5 validation of production of Single-stranded DNA-parainfluenza 2 target Using Universal Single-tailed asymmetric amplification technology
The feasibility of the method was examined with respect to the parainfluenza 2 target according to the reaction system, reaction conditions, etc. in example 1. The results are shown in FIG. 7, and the fluorescent signal appears in FIG. 7, which is clearly distinguished from the blank, and the result is observed under the irradiation of ultraviolet lamp, which shows that the detection of the nucleic acid target is feasible and has good specificity.
The copy number of the DNA template in the PCR system is 10 3 copies/. Mu.L; in the fluorescent signal detection method of this embodiment, the fluorescent signal is collected by a 7300Plus real-time fluorescent quantitative PCR instrument.
Target specific primer sequence (parainfluenza 2):
FLG2-S1-F(SEQ ID No.10)：5’-ACAGCGGAATGGGAGGATTTT-3’(Tm：66.4℃)
FLG2-S1-R (SEQ ID No. 11): 5'-TTTTTAGTTTACATTGTGCATGTACTTGGCCATGGGTCCT-3' (Tm: 67.7 ℃ C.; total Tm:73.9 ℃ C.)
Example 6 validation of Single-stranded DNA-parainfluenza 3 target Using Universal Single-tailed asymmetric amplification technique
The feasibility of the method was examined with respect to the parainfluenza 3 target according to the reaction system, reaction conditions, etc. in example 1. The results are shown in FIG. 8, the fluorescent signal appears in FIG. 8, and is clearly distinguished from the blank, and the results are observed under the irradiation of ultraviolet lamps, which indicates that the detection of the nucleic acid target is feasible and has good specificity.
The copy number of the DNA template in the PCR system is 10 3 copies/. Mu.L; in the fluorescent signal detection method of this embodiment, the fluorescent signal is collected by a 7300Plus real-time fluorescent quantitative PCR instrument.
Target specific primer sequence (parainfluenza 3):
FLG3-S1-F(SEQ ID No.12)：5’-GCGGCATTATACCCATCTGTT-3’(Tm：64.6℃)
FLG3-S1-R (SEQ ID No. 13): 5'-TTTTTAGTTTACATTGTGCGGGACTATGAGATGCCTGATTG-3' (Tm: 64.1 ℃ C.; total Tm:72.6 ℃ C.)
Example 7 validation of Single-stranded DNA-metapneumovirus target Using Universal Single-tailed asymmetric amplification technique
The feasibility of the method was examined for metapneumovirus targets according to the reaction system, reaction conditions, etc. in example 1. The results are shown in FIG. 9, the fluorescent signal appears in FIG. 9, and is clearly distinguished from the blank, and the result is observed under the irradiation of ultraviolet lamp, which shows that the detection of the nucleic acid target is feasible and has good specificity.
The copy number of the DNA template in the PCR system is 10 3 copies/. Mu.L; in the fluorescent signal detection method of this embodiment, the fluorescent signal is collected by a 7300Plus real-time fluorescent quantitative PCR instrument.
Target specific primer sequences (metapneumovirus):
PF-S1-F(SEQ ID No.14)：5’-ACCAGACACACCCATAATCTT-3’(Tm：63.4℃)
PF-S1-R (SEQ ID No. 15): 5'-TTTTTAGTTTACATTGTGCTCACTTAGTACACGGTTAGCCC-3' (Tm: 65.2 ℃ C.; total Tm:72.3 ℃ C.)
Example 8 application of Universal Single-tailed asymmetric amplification technique to Single-stranded DNA production-syncytial Virus target
After verification of single-stranded DNA generation is completed by using a universal single-tailed asymmetric amplification method, a simulated clinical sample of syncytial virus is used in this example to illustrate the application of the nucleic acid detection method set forth in the present invention.
In the simulated clinical samples related to this example, positive samples were all DNA simulated synthetic samples obtained by reverse transcription techniques after dilution with different concentrations of syncytial viral RNA, and negative samples were all enzyme-free water not containing each target DNA. In the application, 6 positive samples and 4 negative samples are adopted.
In the fluorescent signal detection method of this embodiment, the fluorescent signal is collected by a 7300Plus real-time fluorescent quantitative PCR instrument.
The reaction system:
TABLE 4 reverse transcription System design
TABLE 5PCR System design
* Primer final concentration: 10. Mu.M;
TABLE 6Cas-FQ architecture design
Q-PCR conditions: 37-30 s; 37-30 s [ 60cycles ]
PCR reaction conditions:
TABLE 7 PCR temperature program for reverse transcription
TABLE 8PCR temperature program
Sample preparation:
Table 9 positive simulated sample preparation
The reaction process comprises the following steps:
Firstly, preparing a simulated clinical sample, extracting RNA from the prepared target RNA by adopting an RNA extraction reagent, quantifying the RNA to 10 4、105、106 copies/. Mu.L, adding enzyme-free water for random dilution, and completing preparation of a positive simulated sample, wherein the preparation of the positive simulated sample is shown in the table 9; the prepared sample is subjected to reverse transcription technology, the specific steps are shown in the reverse transcription system design of table 4 and the reverse transcription PCR temperature program of table 7, a cDNA template is synthesized, the cDNA template is added into the PCR system shown in table 2, and a DNA simulated positive sample is synthesized through PCR amplification. The simulation of negative samples was also accomplished by the method described above.
The simulated sample is added into a Cas-FQ system, namely, firstly 10 XNEB Buffer 2.1 Buffer solution, cas12a protein, crRNA corresponding to a syncytial virus target, fluorescent probe FQ and water are mixed to complete a premix, then the DNA simulated sample is added, and the reaction is carried out according to qPCR conditions, wherein the design of the Cas-FQ system is shown in the table 6. Three-hole repeated verification is carried out on each sample, the specific result is shown in fig. 10, signals of positive samples are all lifted, fluorescent signals appear, the signals are obviously distinguished from negative samples, and the results are visually observed under the irradiation of ultraviolet lamps, so that 6 of the 10 simulated samples are syncytial virus positive samples.
Example 9 use of Single-tailed Universal asymmetric amplification technique to generate Single-stranded DNA-Equipped viral targets
This example was conducted by using the method for the B-stream virus target according to the reaction system, reaction conditions, etc. in example 8.
In the simulated clinical samples related to the embodiment, positive samples are DNA simulated synthetic samples obtained by reverse transcription technology after being diluted by different concentrations of the B-stream viral RNA, and negative samples are enzyme-free water which does not contain target DNA. In the application, 6 positive samples and 4 negative samples are adopted.
In the fluorescent signal detection method of this embodiment, the fluorescent signal is collected by a 7300Plus real-time fluorescent quantitative PCR instrument.
The results are shown in fig. 11, the signals of the positive samples are all raised, fluorescent signals appear, the signals are obviously distinguished from the negative samples, and the results are visually observed under the irradiation of ultraviolet lamps, so that 6 of the 10 simulated samples are all positive samples of the b-flow virus.
Example 10 application of Single-tailed Universal asymmetric amplification technique to Single-stranded DNA production-metapneumovirus target
This example uses the method according to the reaction system, reaction conditions, etc. in example 8 for a metapneumovirus target.
In the simulated clinical samples related to this example, positive samples were all DNA simulated synthetic samples obtained by reverse transcription techniques after dilution with different concentrations of metapneumoviral RNA, and negative samples were all enzyme-free water not containing each target DNA. In the application, 6 positive samples and 4 negative samples are adopted.
In the fluorescent signal detection method of this embodiment, the fluorescent signal is collected by a 7300Plus real-time fluorescent quantitative PCR instrument.
The results are shown in fig. 12, the signals of the positive samples are all raised, fluorescent signals appear, the signals are obviously distinguished from the negative samples, and the results are visually observed under the irradiation of ultraviolet lamps, so that 6 of the 10 simulated samples are metapneumovirus positive samples.
The results of examples 8-10 show that the single-stranded DNA product amplification method of the invention is convenient to detect, high in amplification efficiency and high in sensitivity when applied to pathogen nucleic acid detection.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Meanwhile, the above embodiments are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereto, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the present invention.
Claims (5)
1. A method for amplifying a single-stranded DNA product, said method comprising the steps of:
Step 1, designing a universal single-tailed primer: screening a segment of non-human genome sequence irrelevant to a detection target sequence in a genome database, and searching a segment of segment from the sequence to serve as a universal single-tailed primer;
step 2, designing a primer pair according to a pathogen nucleic acid sequence;
Step 3, designing target specific primers: connecting the 3 'end of the universal single tailing primer sequence to the 5' end of one pathogen nucleic acid sequence primer sequence;
step 4, PCR amplification: in the first stage, target specific primers are amplified exponentially under annealing temperature conditions; in the second stage, after the exponential amplification is finished, the annealing temperature is reduced, linear amplification is carried out by using a universal single-tailed primer, and finally, a single-stranded DNA product is generated through reaction;
wherein, the Tm value of the universal single-tailed primer is lower than that of the pathogen nucleic acid sequence primer, and the difference between the Tm value and the Tm value is more than or equal to 5 ℃.
2. The method for amplifying a single-stranded DNA product according to claim 1, wherein the sequence length of said universal single-tailed primer is 18-25bp.
3. The method for amplifying a single-stranded DNA product according to claim 2, wherein the sequence of the universal single-tailed primer is: 5'-TTTTTAGTTTACATTGTGC-3'.
4. The method for amplifying a single-stranded DNA product according to claim 1, wherein the PCR amplification system in step 4 is: 5U/. Mu.L Taq enzyme, 0.3. Mu.L; 2.5mM each dNTP,1 μl; target specific primer, 0.3 μl×2; 2 mu L of universal single-tailed primer; 10 XPCR Buffer, 2. Mu.L; ddH 2 O, 12.1. Mu.L; 2. Mu.L of DNA template;
PCR amplification procedure: the first stage: 95 ℃ for 3min; cycling for 30 times at 95 ℃,10s, 65 ℃ and 30 s; and a second stage: 72 ℃,20s; cycling for 20-30 times at 95 ℃,10s, 55 ℃,15s, 72 ℃ and 20s; 72℃for 4min.
5. Use of the method for amplification of single stranded DNA products according to claim 1 for pathogen nucleic acid detection, characterized in that said use is in particular: and combining the single-stranded DNA product amplification method with a CRISPR/Cas system, namely generating a single-stranded DNA product by utilizing the single-stranded DNA product amplification method, combining the single-stranded DNA product with Cas12a and Cr-RNA to form a complex, activating trans-cleavage activity, and cutting fluorescence to generate a signal to finish fluorescence signal detection.
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