CN116287144A - Nucleic acid detection systems, devices and methods - Google Patents
Nucleic acid detection systems, devices and methods Download PDFInfo
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Abstract
The application discloses a nucleic acid detection system, including CRISPR detecting system and nucleic acid amplification system, CRISPR detecting system includes Cas effect protein and does not contain nucleic acid signal molecule, and nucleic acid amplification system includes nucleic acid signal molecule, and CRISPR detecting system and nucleic acid amplification system combine to detect target nucleic acid, and nucleic acid signal molecule is used for reporting detection signal. According to the detection performance verification and stability verification after single-factor material rejection, the CRISPR reagent is found to be free of adding nucleic acid signal molecules, so that the stability of the nucleic acid signal molecules can be ensured, the stability of the CRISPR reagent is improved, and the CRISPR reagent has better detection sensitivity and detection specificity in a fluorescence method and a test strip method.
Description
Technical Field
The present application relates to the field of biological detection technology, and in particular, to a nucleic acid detection system, apparatus and method.
Background
Nucleic acid amplification techniques are classified into two main categories according to the characteristics of temperature cycling dependence: nucleic acid non-isothermal amplification techniques typified by PCR and new generation nucleic acid isothermal amplification techniques. Polymerase chain reaction (polymerase chain reaction, PCR) is a typical representation of nucleic acid amplification techniques that use nucleic acid as a template for replication reactions, typically in a temperature cycler, which provides the desired high, low and medium temperature environment for the PCR reaction. Three phases at a time constitute a cycle. The number of target genes was amplified 1-fold every time the PCR reaction was performed through one cycle. After N cycles, a target gene molecule may be amplified to 2 N Therefore, the target gene of small amount is amplified by exponential amplification to reach the detectable level. PCR technology is often used for detecting, identifying infectious diseases, identifying transgenes, detecting gene mutations, and the like.
Different from the conventional PCR technology, the isothermal nucleic acid amplification technology is characterized in that the exponential amplification of target nucleic acid can be realized under specific temperature conditions, and the efficient amplification of nucleic acid can be realized even under the condition of near room temperature. The whole process of the isothermal amplification reaction of nucleic acid is carried out at the same temperature, the requirement on the instrument is greatly simplified, the reaction time is greatly shortened, and the reaction can be completed by simple and even non-professional equipment such as a heating module, a water bath and the like.
The isothermal amplification products or PCR amplification products of the nucleic acid can be subjected to color development or fluorescence reading by shearing nucleic acid signal molecules through a CRISPR/Cas system, and the nucleic acid can be qualitatively or quantitatively detected. Wherein CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a repetitive sequence in the genome of a prokaryote, which is an immune weapon generated by fight against bacteria and viruses in the history of life evolution, in short, the viruses can integrate own genes into the bacteria, the bacteria can use the cell tools of the bacteria to serve the own gene replication, the bacteria evolve a CRISPR-Cas system for removing the foreign invasive genes of the viruses, and the Casase can directionally search target nucleic acid sequences under the guidance of a segment of RNA by using the CRISPR-Cas system, and then the sequences are excised. The target sequence is excised and the nucleic acid signal molecule is also cleaved to release fluorescence or to undergo a chromogenic reaction.
However, due to the instability of the nucleic acid signal molecules, the detection reagent preservation requirements of the CRISPR/Cas system are high, requiring overall preservation at-20 ℃.
Therefore, how to increase the stability of CRISPR detection reagents is a difficulty in its improvement.
Disclosure of Invention
In order to solve the above problems and improve the stability of CRISPR detection reagents, a first object of the present application is to provide a nucleic acid detection system comprising a CRISPR detection system comprising a Cas effect protein and no nucleic acid signal molecule and a nucleic acid amplification system comprising a nucleic acid signal molecule for reporting a detection signal, the CRISPR detection system and the nucleic acid amplification system being used in combination to detect a target nucleic acid.
According to the detection performance verification and stability verification after single-factor material rejection, the CRISPR detection reagent is found to be free of adding nucleic acid signal molecules, so that the stability of the nucleic acid signal molecules can be ensured, the stability of the CRISPR detection reagent is improved, and the CRISPR detection performance is improved, and the CRISPR detection reagent has better detection sensitivity and detection specificity in a fluorescence method and a test strip method.
In one embodiment, the CRISPR detection system further comprises a guide RNA for binding to a corresponding target nucleic acid.
In one embodiment, the nucleic acid amplification system further comprises a guide RNA for binding to a corresponding target nucleic acid.
In one embodiment, the target nucleic acid is a target RNA and the CRISPR detection system further comprises an RNA polymerase.
In one embodiment, the RNA polymerase comprises at least one of T7 RNA polymerase, sp6 RNA polymerase.
In one embodiment, the nucleic acid amplification system further comprises amplification primers with RNA polymerase recognition sites.
In one embodiment, the Cas effector protein is an RNA targeting effector protein.
In one embodiment, the RNA targeting effector protein is Cas13a and/or Cas13b.
In one embodiment, the nucleic acid signal molecule comprises RNA, and the detection signal is generated based on cleavage of the RNA.
In one embodiment, the CRISPR detection system further comprises rtps, rnase inhibitors, mg 2+ And HEPES.
In one embodiment, the working concentration of each component in the CRISPR detection system meets at least one of the following features (1) - (8):
(1) Working concentration of gRNA is 5nM-50
M；
(2) The working concentration of the Cas effect protein is 5nM-500nM;
(3) The working concentration of RNA polymerase is 0.5U-10U;
(4) The working concentration of rNTPs is 1mM-8mM;
(5) The working concentration of the nucleic acid signal molecules is 1nM-2000nM;
(6) The working concentration of the RNase inhibitor is 0.5U-10U;
（7）Mg 2+ the working concentration of (2) is 5mM-20mM;
(8) HEPES works at a concentration of 5mM-20mM.
In one embodiment, the target nucleic acid is a target DNA.
In one embodiment, the Cas effector protein is a DNA targeting effector protein.
In one embodiment, the DNA targeting effector protein comprises Cas9 and/or Cas12, and Cas12 comprises at least one of Cas12a, cas12b, cas12 c.
In one embodiment, the nucleic acid signal molecule comprises DNA, and the detection signal is generated based on cleavage of the DNA.
In one embodiment, the CRISPR detection system further comprises dNTPs, mg 2+ And HEPES.
In one embodiment, the working concentration of each component in the CRISPR detection system meets at least one of the following features (1) - (6):
(1) Working concentration of gRNA is 5nM-50
M；
(2) The working concentration of the Cas effect protein is 5nM-500nM;
(3) The working concentration of the nucleic acid signal molecules is 1nM-2000nM;
（4）Mg 2+ the working concentration of (2) is 5mM-20mM;
(5) HEPES works at a concentration of 5mM-20mM;
(6) The working concentration of each base in dNTPs is 1mM-8mM.
In one embodiment, the nucleic acid amplification system is used to perform any one of the following amplification methods:
recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, rolling circle amplification, cross primer isothermal amplification, strand displacement amplification, helicase dependent amplification, and PCR amplification.
In one embodiment, the nucleic acid amplification system and/or CRISPR detection system further comprises a lyoprotectant.
A second object of the present application is to provide a nucleic acid diagnostic device comprising one or more independent modules, each comprising the above-described nucleic acid detection system.
In one embodiment, the stand-alone module comprises one or more containment units, each provided with a nucleic acid amplification system and/or a CRISPR detection system in a nucleic acid detection system.
In one of the embodiments, the containing unit is for containing at least one of a liquid and a solid, preferably a solid.
In one embodiment, the reagents contained in the nucleic acid amplification system and/or the CRISPR detection system are solid.
In one embodiment, the solid comprises any one of lyophilized microspheres, lyophilized cake, lyophilized powder, and spots depending on the presence of the solid medium.
A third object of the present application is to provide a method for detecting a target nucleic acid in a sample, which is contacted with the above-described nucleic acid detection system for the reaction to determine the target nucleic acid in the sample.
A fourth object of the present application is to provide a method for detecting a target nucleic acid in a sample, comprising the steps of:
(1) Adding a sample or a sample set to the nucleic acid diagnostic device, and contacting the nucleic acid amplification system and the CRISPR detection system in the containment unit to generate a detectable signal;
(2) Detecting the detectable signal, and determining one or more target nucleic acids in the sample.
In one embodiment, the nucleic acid amplification system is used to perform any one of the following methods:
recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, rolling circle amplification, cross primer isothermal amplification, strand displacement amplification, helicase dependent amplification, and PCR amplification.
In one embodiment, the target nucleic acid in the sample is determined using fluorescence or lateral flow immunochromatography;
optionally, the lateral flow immunochromatography performs at least one of a line display method and a line elimination method.
A fifth object of the present application is to provide a method for preparing a nucleic acid detection kit, comprising:
And mixing the nucleic acid amplification system and the CRISPR detection system in the nucleic acid detection system with a freeze-drying protective agent respectively to obtain a mixture, and performing freeze-drying treatment on the mixture.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of a novel RAA-CRISPR assay system of example 5 of the present application stored at-20deg.C;
FIG. 2 is a graph showing the results of a conventional RAA-CRISPR assay system stored at-20℃in example 5 of the present application.
Detailed Description
Reference now will be made in detail to the embodiments of the application, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the present application. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the scope or spirit of the present application. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Accordingly, it is intended that the present application cover such modifications and variations as fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present application are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present application.
As above, the present application finds that conventional CRISPR test reagents are less stable, by conventional test procedures, such as gradient validation of the various materials, and at the same time verify that the stability of the CRISPR test reagents is not improved after adjustment. In addition, the CRISPR test detection by the fluorescence method has a problem of high reaction background noise and occasional occurrence of false negative.
To solve the above technical problem, a first aspect of the present application provides a nucleic acid detection system comprising a CRISPR detection system comprising a Cas effect protein and no nucleic acid signal molecule and a nucleic acid amplification system comprising a nucleic acid signal molecule for reporting a detection signal, the CRISPR detection system and the nucleic acid amplification system being used in combination to detect a target nucleic acid.
Conventional CRISPR comprises two parts: a guide RNA (gRNA) and a nonspecific CRISPR-binding endonuclease (CRISPR-associated endonumclease, cas enzyme). In this application, the term "Cas enzyme" is interchangeable with Cas effector proteins. Cas effector proteins include at least one of Cas12a, cas12b, cas12c, cas13a, cas13b, cas 14.
The CRISPR-Cas12 system recognizes the dsDNA containing PAM under the guidance of the guide RNA, then promotes the target dsDNA to be melted, and a Target Strand (TS) in the melted target dsDNA forms Rloop with the guide RNA, so that the active site of RuvC in the Cas12 protein is released; whereas the non-target strand (NTS) in the target dsDNA immediately after melting is now cleaved by RuvC of the released active site; the result of this cleavage causes the target DNA to unwind, the TS of released target dsDNA being cleaved by RuvC; when Cas12 completes cleavage of TS and NTS in the target dsDNA (cis-cleavage), dsDNA is released, and the active site of RuvC, where space is left, is cleaved (trans-cleavage) once ssDNA is entered.
Cas12 Sup>A and Cas12b proteins are subtypes in Cas protein Class 2V, V-Sup>A Cas12 Sup>A, also known as cpf1; V-B cas12B, also known as c2c1. The main differences between Cas12b and Cas12a are: nuclease domains differ: the domain of Cas12b protein is RuvC; the domains of Cas12a proteins are RuvC and Nuc; guide RNAs are different: cas12b requires crRNA and tracrRNA; cas12a requires only crRNA, not tracrRNA; the enzyme cutting modes are different: cas12b cleaves 7 staggered nucleotides; cas12a cleaves staggered ends, 5' overhangs; the application technology is different: a technique for Cas12b protein application is HOLMES v2; techniques applied to Cas12a protein are detect and HOLMES.
After the CRISPR-Cas13 system is combined with crRNA and a substrate to form a ternary complex, the Cas13 protein is activated, and at the moment, the Cas13 protein can cut not only the substrate RNA but also any single-stranded RNA free in the environment. Cas13a proteins and Cas13b proteins are rnases and detection based on Cas13a and Cas13b requires a transcription step of the RNA polymerase.
Cas13a needs to interact with Cas13a molecules through uracil-rich stem loop structures and promote cleavage of the target through a series of conformational changes of Cas13 a. Cas13a can tolerate a single mismatch between the crRNA and the target sequence, but if there are 2 mismatches, then the cleavage efficiency is greatly reduced. Its PFS sequence (corresponding to PAM sequence) is located 3' to the spacer and consists of A, U or C bases. Once Cas13a recognizes and cleaves the RNA target specified by the crRNA sequence, it shifts to an enzymatic "activated" state, where it will bind and cleave other RNAs, whether or not they are homologous to the crRNA or whether PFS is present. This property is of interest for bacteria. If the bacterium is infected with a phage, it can activate a programmed cell death or dormant state to limit the spread of the infection throughout the population.
Like Cas13a, cas13b requires only a single guide RNA to find the target and is encodable from a genetic perspective. Furthermore, cas13b is able to target multiple RNA transcripts simultaneously. But Cas13b also has some unique features that suggest it is different from Cas13a, which make Cas13b more suitable for trimming gene functions.
Cas14 nuclease is an endonuclease that specifically binds and cleaves target ssDNA under the guidance of a tracrRNA: crRNA (or sgRNA), and does not require a PAM site. Cas14 proteins are generally smaller in molecular weight (400-700 aa) compared to other Cas proteins; similar to Cas12, cas14 can also bind to target nucleic acids and activate its ssDNA trans-cleavage activity, thereby being applied to molecular detection of target nucleic acids; unlike Cas12, cas14 only binds to ssDNA targets, thus amplified enriched target nucleic acids need to be treated with T7 exonuclease, and one of the amplification primers needs to be modified with phosphorothioate to ensure that T7 exonuclease cleaves only one strand, leaving ssDNA target strands for Cas 14-mediated molecular detection.
The guide RNA, that is, the gRNA, is also called sgRNA. gRNA is manufactured artificially by humans and is not found in nature. Such gRNA designs are based on naturally occurring crrnas and/or tracrrnas. For example, the addition of a GAAA linker between the two fragments of naturally occurring crRNA and naturally occurring tracrrRNA can make them gRNA fragments.
In order to detect the targeting sequence, the conventional CRISPR reagent further comprises a nucleic acid signal molecule, wherein the nucleic acid signal molecule comprises a segment of nucleic acid, a fluorescent group linked with the nucleic acid and a quenching group (or a similar arrangement), and the Cas enzyme cuts off the nucleic acid chain between the fluorescent group and the quenching group after cutting the specific nucleic acid target once, so that the nucleic acid signal molecule emits signals such as fluorescence. Depending on the Cas effector protein, the nucleic acid signal molecule may be a signal reporter RNA or a signal reporter DNA.
According to the CRISPR detection reagent, through detection performance verification and stability verification after single-factor material rejection, the CRISPR detection reagent is found to be free of adding nucleic acid signal molecules, so that stability of the nucleic acid signal molecules can be guaranteed, stability of the CRISPR detection reagent can be improved, and the CRISPR detection reagent has better detection sensitivity and detection specificity in a fluorescence method and a test strip method.
The nucleic acid amplification system for amplifying the target nucleic acid may be an isothermal amplification system or a non-isothermal amplification system.
In some embodiments, the nucleic acid amplification system is used to perform any one of the following amplification methods:
recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, rolling circle amplification, cross primer isothermal amplification, strand displacement amplification, helicase dependent amplification, and PCR amplification.
The isothermal amplification (recombinase polymerase amplification, RAA) of recombinase polymerase is to scan double-stranded DNA by forming a complex of recombinase, single-stranded binding protein and primer, unwind the double-stranded DNA at the sequence homologous to the primer, prevent the single-stranded DNA from renaturation by single-stranded binding protein (SSB), and complete the chain extension by DNA polymerase in the presence of energy and dNTPs, thus realizing the instrumental amplification in 5-20 minutes.
Loop-mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) designs four primers for six regions on a target gene, and performs an amplification reaction by using a strand displacement type DNA polymerase under a constant temperature condition, so that 10 can be realized within 15-60 minutes 9 -10 10 Amplification of multiple, reverseThe presence or absence of the target gene can be judged by visually observing the presence or absence of white precipitate, which is a large amount of amplification product, magnesium pyrophosphate. Besides high specificity and high sensitivity, LAMP is also very simple to operate, has low requirements on instruments in the application stage, can realize the reaction by a simple constant temperature device, has very simple detection result, and can be used for directly observing white sediment or green fluorescence by naked eyes.
Rolling circle amplification (rolling circle amplification, RCA) is performed by addition of a restriction enzyme to the circle probe, through one or more DNA primers (complementary to part of the circular template), transforming dNTPs into single stranded DNA containing hundreds to thousands of repeated template complementary fragments under the catalysis of phi29 DNA polymerase.
The cross primer isothermal amplification (CrossingPriming Amplification) is an in vitro isothermal amplification method of DNA based on enzymatic reaction, and is mainly carried out under isothermal conditions by utilizing the capability of restriction enzymes to cut DNA recognition sites and the capability of DNA polymerase to extend 3' at a cut and replace downstream sequences.
Strand displacement amplification (Strand displacement amplification) uses mainly the ability of a restriction enzyme to cleave a DNA recognition site and the ability of a DNA polymerase to extend 3' at a nick and displace downstream sequences, and amplification is performed under isothermal conditions.
Helicase-dependent amplification (helicase-dependent amplification), in which the helicase is used to cleave the DNA duplex at constant temperature, and the cleaved single strand is stabilized by DNA single strand binding protein to provide template for the primer, and complementary duplex is synthesized by DNA polymerase, and the cycle amplification process is repeated.
Thus, it will be appreciated that the nucleic acid amplification reagents used to amplify the target nucleic acid in the nucleic acid amplification system may be isothermal amplification reagents or non-isothermal amplification reagents.
In some embodiments, the CRISPR detection system further comprises a guide RNA for binding to a corresponding target nucleic acid.
Furthermore, the application also finds that the gRNA and the nucleic acid signal molecules are separated from the CRISPR reagent at the same time, so that the nucleic acid amplification-CRISPR detection system has better detection performance and stability. Thus, in other embodiments, the nucleic acid signal molecules and grnas are tuned into the nucleic acid amplification reagents such that the nucleic acid amplification system further comprises guide RNAs for binding to the respective target nucleic acids.
The application finds that the same detection sensitivity can be realized by removing the nucleic acid signal molecules from the CRISPR detection system and adopting fewer CRISPR reagent raw materials, and the screening requirements of the guide RNA are not strict as those of the conventional CRISPR detection system, so that the construction of the nucleic acid amplification-CRISPR detection system is easier to complete.
In some embodiments, the target nucleic acid is a target RNA and the CRISPR detection system further comprises an RNA polymerase for effecting transcription of the amplified double stranded DNA into the target RNA to activate subsequent cas cleavage activity.
In some embodiments, the RNA polymerase comprises at least one of T7 RNA polymerase, sp6 RNA polymerase.
In some embodiments, the nucleic acid amplification system further comprises amplification primers with RNA polymerase recognition sites.
In some embodiments, the Cas effector protein is an RNA targeting effector protein.
In some embodiments, the RNA targeting effector protein is Cas13a and/or Cas13b.
In some embodiments, the nucleic acid signal molecule comprises RNA, and the detection signal is generated based on cleavage of the RNA.
In some embodiments, the CRISPR detection system further comprises rtps, mg 2+ At least one of HEPES and RNase inhibitor.
In some embodiments, the working concentration of each component in the CRISPR detection system meets at least one of the following features (1) - (8):
(1) Working concentration of gRNA is 5nM-50
M；
(2) The working concentration of the Cas effect protein is 5nM-500nM;
(3) The working concentration of RNA polymerase is 0.5U-10U;
(4) The working concentration of rNTPs is 1mM-8mM;
(5) The working concentration of the nucleic acid signal molecules is 1nM-2000nM;
(6) The working concentration of the RNase inhibitor is 0.5U-10U;
（7）Mg 2+ the working concentration of (2) is 5mM-20mM;
(8) HEPES works at a concentration of 5mM-20mM.
In some embodiments, the target nucleic acid is a target DNA.
In some embodiments, the Cas effector protein is a DNA targeting effector protein.
In some embodiments, the DNA targeting effector protein comprises Cas12 and/or Cas14, with Cas12 comprising at least one of Cas12a, cas12b, cas12 c.
In some embodiments, the nucleic acid signal molecule comprises DNA, and the detection signal is generated based on cleavage of the DNA.
In some embodiments, the CRISPR detection system further comprises dNTPs, mg 2+ And HEPES.
In some embodiments, the working concentration of each component in the CRISPR detection system meets at least one of the following features (1) - (6):
(1) Working concentration of gRNA is 5nM-50
M；
(2) The working concentration of the Cas effect protein is 5nM-500nM;
(3) The working concentration of the nucleic acid signal molecules is 1nM-2000nM;
（4）Mg 2+ the working concentration of (2) is 5mM-20mM;
(5) HEPES works at a concentration of 5mM-20mM;
(6) The working concentration of each base in dNTPs is 1mM-8mM.
The nucleic acid amplification reagent for amplifying the target nucleic acid may be an isothermal amplification reagent or a non-isothermal amplification reagent. In some embodiments, to improve the long-term storage stability of the nucleic acid detection system, the nucleic acid amplification system and/or the CRISPR detection system further comprise a lyoprotectant to facilitate preparation, transport, and storage of the nucleic acid detection system.
In a second aspect, the present application provides a nucleic acid diagnostic device comprising one or more independent modules, each comprising a nucleic acid detection system as described above, different independent modules being capable of detecting different target nucleic acids.
In some embodiments, the stand-alone module comprises one or more containment units, each provided with a nucleic acid amplification system and/or a CRISPR detection system in a nucleic acid detection system.
In some embodiments, the containment unit is for containing at least one of a liquid and a solid, preferably a solid.
In some embodiments, the reagents contained in the nucleic acid amplification system and/or the CRISPR detection system are solid.
In some embodiments, the solid comprises any of lyophilized microspheres, lyophilized cake, lyophilized powder, spots depending on the presence of the solid medium.
In a third aspect the present application provides a method for detecting a target nucleic acid in a sample, the sample being contacted with the nucleic acid detection system described above to determine the target nucleic acid in the sample.
In a fourth aspect the present application provides a method for detecting a target nucleic acid in a sample, comprising the steps of:
(1) Adding a sample or a sample set to the nucleic acid diagnostic device, and contacting the nucleic acid amplification system and the CRISPR detection system in the containment unit to generate a detectable signal;
(2) Detecting the detectable signal, and determining one or more target nucleic acids in the sample.
In the above method of detecting a target nucleic acid in a sample, the nucleic acid amplification system is used to perform any one of the following methods:
Recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, rolling circle amplification, cross primer isothermal amplification, strand displacement amplification, helicase dependent amplification, and PCR amplification.
In the above method for detecting a target nucleic acid in a sample, the target nucleic acid in the sample is measured by fluorescence or lateral flow immunochromatography;
optionally, the lateral flow immunochromatography performs at least one of a line display method and a line elimination method.
According to the detection requirements, the detection method of the present application can detect target nucleic acids extracted from foods, sewage, animal body fluids, and the like.
In some embodiments, the target nucleic acid comprises at least one of a monkey poxvirus nucleic acid and a novel coronavirus nucleic acid.
In a fifth aspect, the present application provides a method for preparing a nucleic acid detection kit, comprising:
the nucleic acid amplification system and the CRISPR detection system in the nucleic acid detection system are respectively mixed with the freeze-drying protective agent to obtain a mixture, and the mixture is subjected to freeze-drying treatment, so that the long-term stability of the freeze-dried microspheres is improved, and the industrialized production of the kit is realized.
In some embodiments, lyophilizing the nucleic acid amplification lyophilization reagents and CRISPR lyophilization reagents specifically comprises:
Pre-cooling the nucleic acid amplification freeze-drying reagent and the CRISPR freeze-drying reagent;
heating according to a preset temperature gradient within the range of-50 ℃ to 20 ℃, and vacuum drying the precooled nucleic acid amplification freeze-dried reagent and the CRISPR freeze-dried reagent for 19 hours to obtain the nucleic acid amplification reagent and the CRISPR reagent which can keep long-term stability.
Embodiments of the present application will be described in detail below with reference to examples, but the present application is not limited to these examples. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 conventional isothermal amplification-CRISPR detection System
1.1 preparation of samples
1.1.1 Fragment sequence to be detected
(1) The monkey poxvirus F3L gene and 2019-nCoV novel coronavirus N gene specific target gene fragments are designed by referring to NCBI sequences MT250197.1 and NC_045512 and are synthesized by general biology (Anhui) stock, and are specifically shown in table 1.
TABLE 1
1.1.2 Preparation of pseudovirus sample sequences
1) Preparation of monkey pox F3L pseudovirus:
in the embodiment, the monkey pox F3L pseudovirus obtains a Monkey pox Virus-F3L gene sequence by a chemical synthesis method, clones and constructs an Ad5 replication defective adenovirus vector, prepares the pseudovirus in 293A cells, and purifies the obtained pseudovirus by a chromatographic column to obtain a DNA sequence wrapped by an adenovirus capsid.
2) Preparation of 2019-nCoV novel coronavirus N-gene pseudovirus:
the novel coronavirus N gene sequence is synthesized and cloned and constructed to a lentiviral vector, and pseudoviruses are prepared in 293T cells, and the pseudoviruses are RNA sequences containing N genes in lentiviral genomes.
1.1.3 Nucleic acid extraction of pseudovirus samples
The method uses an Anhui as a reagent kit for extracting and purifying nucleic acid of a biological engineering technology Co., ltd to extract a pseudo-virus sample, and the reagent kit can be compatible for extracting DNA and RNA of the virus sample, so that the nucleic acid extraction of two viruses can be completed by the same extraction flow for amplification and detection.
The nucleic acid extraction procedure was as follows:
(1) 300 was added to a 1.5mL centrifuge tube
Sample releasing agent of L, add 160
And L isopropanol and shaking and mixing uniformly.
(2) 200 mu L of sample is added, vortex vibration is carried out for 30s, and vibration and mixing are carried out uniformly.
(3) Adding 20 to the sample
And (3) mixing the L nucleic acid extracting agent by shaking, reversing and uniformly mixing on a rotary mixer for 5min, sucking the mixed liquid into a 1.5mL centrifuge tube after instantaneous separation, placing the centrifuge tube on a magnetic rack, adsorbing for 60s, and discarding the supernatant.
(4) 500 mu L of cleaning solution (confirming that ethanol is added) is added into the centrifuge tube, the magnetic beads are resuspended, the magnetic beads are vigorously vibrated for 30s, the magnetic beads are placed on a 1.5mL magnetic rack after being instantaneously separated, the magnetic beads are adsorbed for 60s, and the supernatant is discarded.
(5) 500 mu L of 85% ethanol solution is added into a centrifuge tube, vortex is carried out for 30s, the mixture is placed on a 1.5mL magnetic rack after instantaneous separation, the mixture is adsorbed for 60s, and the supernatant is discarded.
(6) The operation (5) was repeated 1 time.
(7) Taking down the centrifuge tube, placing the surface with the magnetic beads attached to the outside in the centrifuge, and rapidly separating the magnetic beads at a high speed.
(8) And placing the centrifuge tube on a magnetic frame, sucking residual liquid by using a gun head, uncovering a metal bath at 56 ℃, airing for 2-5 minutes, and airing until the surface of the magnetic bead is frosted. Adding 20-100 mu L of elution buffer preheated at 56 ℃, resuspending by using a pipette, and standing for 2 minutes at room temperature.
(9) After transient separation, the sample was placed on a 1.5mL magnetic rack, adsorbed for 60s, and the supernatant was aspirated into a new RNase Free centrifuge tube.
The extracted nucleic acid can be directly used for subsequent amplification.
1.2 isothermal amplification
(1) The RAA recombinase polymerase amplification reagent contains DNA polymerase 5-10U, T phage UvsX recombinase 1-4
g. Single-chain Binding protein (SSB) 5
g~10
g. Recombinant enzyme binding factor UvsY 0.5
g~1
g. A pair of long primers with a length of about 30-35 bases and Mg 2+ dNTPs, DTT, primers, buffers, and the like, and when the detection template is RNA, reverse transcriptase is also included. The RAA reaction temperature is 37-42 ℃, and the reaction time is 20min.
(2) The LAMP loop-mediated isothermal amplification reagent comprises 4 primers (2 outer primers and 2 inner primers) and a strand displacement DNA polymerase (Bst DNA polymerase) 2U-10U, dNTPs, template DNA, betaine and Mg 2 SO 4 Amplification primers, and the like, and reverse transcriptase is also included when the detection template is RNA. The LAMP reaction conditions were 40min at 65 ℃.
(3) RCA rolling circle amplification reagent comprises Ase I restriction enzyme 5U-10U, T4 DNA ligase DNA 3U-7U, phi29 DNA polymerase 5U-10U, amplification primer and the like, and reverse transcriptase is also included when the detection template is RNA. RCA reaction conditions were amplification for 5 hours at about 30 ℃.
(4) CPA cross primer amplification reagent comprises 10U-20U of large DNA polymerase fragment of bacillus stearothermophilus, and the dosage of 5-10 mol/L betaine is 2
L, dNTPs amplification primers, and the like, and reverse transcriptase is also included when the detection template is RNA. CPA reaction temperature is 60 ℃ and reaction time is 60min.
(5) The SDA strand displacement amplification reagent comprises BsoBI 1-5U/L, bst DNA Polymerase U-10U, T4 polynucleotide kinase 2U-5U, 2 '-deoxydenosine 5' -O- (1-thiophosphorate), dNTPs, BSA, DMSO, an amplification primer, reverse transcriptase and the like when the detection template is RNA. SDA reaction conditions were 30min at 37 ℃.
(6) The HDA helicase dependent amplification reagents were used in amounts according to IsoAmp ii Universal tHDA Kit, purchased from NEB. The main component is MgSO 4 3.5-4 mM, 30-40 NaCl mM, protoScript II RT 1-2U, dNTPs, amplification primers, reverse transcriptase when the detection template is RNA, and the like.
Primer sequences required for isothermal amplification are shown in table 2 below.
TABLE 2
1.3 CRISPR detection system
CRISPR detection systems recognize target nucleic acids and activate bypass-cleaving active cleavage reporter probes under the direction of sequence-specific RNA molecules. The specific recognition and detection of the DNA target product and the RNA target product can be accomplished by selecting different Cas enzymes.
(1) Detection of target DNA:
cas active proteins such as Cas12a enzyme, cas12b enzyme, cas12c enzyme, cas14 enzyme, etc., can be identified by targeted complementary DNA sequences through guide RNAs. Upon binding to the target DNA, the Cas active protein induces cleavage in the target DNA strand while also inducing trans-cleavage of non-target DNA, generating a fluorescent signal or chromogenic detection by a dipstick. Cas active proteins such as Cas12a enzyme, cas12b enzyme and the like can directly identify and cleave Wen Kuozeng products, and reaction components comprise gRNA 10nM-50
M, cas12a/b protein 10-500nM, HEPES 5-20mM, mgCl 2 5-20mM, 2-2000nM fluorescent probe, template, etc. For Cas12a enzymes, the gRNA is the crRNA alone, and for Cas12b enzymes, the gRNA is a complex of crRNA and tracrRNA.
(1) Fluorescence method: the detection system is placed in a qPCR instrument (taking a report probe as FAM-BHQ1 as an example, the excitation light wavelength is 490 and nm, the emission light wavelength is 520 and nm, the reaction temperature is 37 ℃, fluorescence is collected every 1 min for 35 times), the fluorescence intensity change in the detection system is recorded, and the fluorescence signal intensity of an end point is recorded.
(2) Test strip method: and (3) placing the detection system in a thermostat to incubate for 20min at 37 ℃, and then dipping the detection system with a test strip or immersing a sample pad in the reaction system to detect the test strip.
(2) Detection of target RNA:
cas13a enzymes can target recognition of complementary RNA sequences by guide RNAs. At the same time withAfter target RNA binding, cas13a protein induces cleavage in the target RNA strand while also inducing cleavage of non-target RNA, producing a fluorescent signal or a test strip color development. The product to be identified by the Cas13a enzyme is RNA, the isothermal amplification product needs to be transcribed to reach the detection requirement, and the main reaction components comprise gRNA10nM-50
M, cas13a protein 10-500nM, RNase inhibitor 1-10U, T RNA polymerase 1-10U, rNTPs 2-8mM, mg 2+ 5-20mM, HEPES 5-20mM, fluorescent probe 2-2000nM, template, etc.
(1) Fluorescence method: the detection system is placed in a qPCR instrument (taking a report probe as FAM-BHQ1 as an example, the excitation light wavelength is 490nm, the emission light wavelength is 520nm, the reaction temperature is 37 ℃, fluorescence is collected every 1 min for 35 times, and the fluorescence intensity change in the detection system is recorded, and the fluorescence signal intensity of an end point is recorded.
(2) Test strip method: and (3) placing the detection system in a thermostat to incubate for 20min at 37 ℃ and then loading and detecting the test strip.
Oligonucleotide sequences used for the CRISPR different Cas enzyme systems are shown in table 3 below:
TABLE 3 Table 3
Example 2 novel RAA-CRISPR detection System suitable for DNA target detection
In this example, a single factor test was performed to confirm the components affecting the stability of the CRISPR detection system, taking the Cas12a/b detection system as an example. In this example, the monkey poxvirus F3L gene SEQ ID NO:1 detection system a correlation study was performed.
Study method:
1. primer SEQ ID NO:3/SEQ ID NO:4 amplifying the simian poxvirus F3L pseudovirus extract, wherein the amplified loading amount is 10copy/T and 1copy/T.
2. Cas12a, cas12b, specific gRNA, in particular SEQ ID NO:17/SEQ ID NO: and 18, configuring a CRISPR detection system. Wherein, for Cas12a enzyme, the gRNA is a separate crRNA, and for Cas12b enzyme, the gRNA is a complex of crRNA and tracrRNA.
The main components of the novel CRISPR detection system suitable for DNA target detection comprise gRNA 5nM-50
M, cas12a/b protein 5-500nM, HEPES 5-20mM, mgCl 2 5-20mM, 1-2000nM reporter probe, template, etc.
Conventional CRISPR detection systems include reporter probes of 10nM-50
M, gRNA 5-5000nM, cas enzyme (Cas 12a/Cas12 b) 10-500nM, mgCl 2 5-20mM, HEPES 5-20mM. In this example, a conventional CRISPR assay system, specifically the composition shown in table 5, was used as a control; further, the reporter probe, the gRNA, the Cas enzyme (Cas 12a/Cas12 b), and the MgCl are respectively used 2 HEPES was removed from the conventional CRISPR detection system, specifically panels 1-5 of Table 5. Wherein groups 1-5 and the control group were tested after 3 days of storage at 25 ℃.
Verification of crispr detection system effect stability component the system formulations are shown in tables 4 and 5:
TABLE 4 Table 4
TABLE 5
Pre-amplification was performed according to the isothermal amplification method of RAA in example 1, followed by 5
The amplified product of LRAA is added into a CRISPR detection system, and is detected by adopting a fluorescence method and a test strip method.
(II) detection results
CRISPR-Cas12a/b detection System
(1) Fluorescence detection result
The detection system of this example was placed in a qPCR apparatus (excitation light wavelength 490 nm, emission light wavelength 520 nm, reaction temperature 37 ℃ C.; fluorescence was collected every 1 min for 35 times total), and the fluorescence intensity change in the detection system was recorded.
The fluorescence detection results of CRISPR-Cas12a are shown in table 6:
TABLE 6
CRISPR-Cas12b detection results are shown in table 7:
TABLE 7
Analysis of results:
(1) conventional CRISPR detection systems are stable in storage at-20 ℃;
(2) the conventional CRISPR detection system is unstable in storage at 25 ℃ and is not beneficial to the industrialized production of reagents;
(3) in the novel CRISPR detection system (group 1), the reaction system of RAA and CRISPR (without a report probe) can be stably stored for 3 days at 25 ℃, is consistent with the storage lower limit of-20 ℃ and has higher fluorescence value, thereby being convenient for daily operation and transportation and meeting the freeze-drying requirement.
(2) Test result-line display method by test paper strip method
After the above detection system of this example was incubated at 37℃for 20min with a thermostat, test strips (purchased from Shanghai Liangrun Biotechnology Co., ltd.) were loaded for detection.
The Cas12a strip-line assay results are shown in table 8:
TABLE 8
The Cas12b test strip line-up detection results are shown in table 9:
TABLE 9
Note that: the method is line display detection, T is a detection site, and C is a quality control site. T+ represents that the product detects the report probe, and the result is positive; t-represents no reporter probe detected in the product and the result is negative. Negative. C+ represents qualified quality control, and the result is effective; c-represents unqualified quality control, and results are invalid.
Analysis of results:
(1) conventional CRISPR detection systems are stable in storage at-20 ℃;
(2) the conventional CRISPR detection system is unstable in storage at 25 ℃ and is not beneficial to the industrialized production of reagents;
(3) in the novel CRISPR detection system (group 1), the reaction system of RAA and CRISPR (without a reporter probe) can be stably stored for 3 days at 25 ℃, is consistent with the storage lower limit of-20 ℃, and is convenient for daily operation and transportation and meets the freeze-drying requirement.
(3) Test result by test paper strip method-line eliminating method
The test results of the Cas12a test strip vanishing method are shown in table 10:
table 10
The test results of the Cas12b test strip vanishing method are shown in table 11:
TABLE 11
Note that: the method is a line elimination method detection, T is a detection site, and C is a quality control site. T+ represents that the report in the product is not completely degraded by Cas12a/b, and the result is negative; the T-representative product reported complete degradation by Cas12a/b, and was positive. C+ represents qualified quality control, and the result is effective; c-represents unqualified quality control, and results are invalid.
Analysis of results:
(1) conventional CRISPR detection systems are stable in storage at-20 ℃;
(2) the conventional CRISPR detection system is unstable in storage at 25 ℃ and is not beneficial to the industrialized production of reagents;
(3) in the novel CRISPR detection system (group 1), the reaction system of RAA and CRISPR (without a reporter probe) can be stably stored for 3 days at 25 ℃, is consistent with the storage lower limit of-20 ℃, and is convenient for daily operation and transportation and meets the freeze-drying requirement.
In summary, the report probe is added into the RAA reaction system from the CRISPR detection system, so that the stability and detection performance of the CRISPR detection system can be improved. The detection of low copy samples has a remarkable effect. Meanwhile, the stability of the CRISPR reagent is increased, the preservation time of the CRISPR reagent is prolonged, and the industrial production is facilitated.
Example 3 novel RAA-CRISPR detection System suitable for RNA target detection
In this example, the Cas13a detection system is taken as an example, and single factor verification is performed to confirm the components affecting the stability of the CRISPR detection system. In this example, the novel coronavirus N gene SEQ ID NO:2 the detection system was subjected to a correlation study.
The research method comprises the following steps:
1) Designing a primer SEQ ID NO: 5. SEQ ID NO:6, the amplified loading was 10copy/T,1copy/T.
2) And (3) designing Cas13a gRNA and configuring a CRISPR detection system.
The novel CRISPR detection system suitable for RNA target detection comprises the following main reaction components: gRNA 1-5000nM, cas13a protein 5nM-50
M, RNA enzyme inhibitor 0.5-10U, T RNA polymerase 0.5-10U, rNTPs 1-8mM, mg 2+ 5-20mM, HEPES 5-20mM, reporter probe 1-2000nM, template, etc.
A conventional CRISPR detection system comprises gRNA 10nM-50
M, cas13a protein 10-500nM, RNase inhibitor 1-10U, T RNA polymerase 1-10U, rNTPs 2-8mM、Mg 2+ 5-20mM, HEPES 5-20mM, fluorescent probe 2-2000nM, template.
In this example, a conventional CRISPR assay system, specifically the composition shown in table 12, was used as a control; further separately separating the reporter probe, gRNA, rNTP, RNA enzyme inhibitor, cas enzyme (Cas 13 a), T7 enzyme, mgCl 2 HEPES was removed from the conventional CRISPR assay system, specifically groups 1-8 of Table 12, wherein groups 1-8 and the control group were tested after 3 days of storage at 25 ℃.
3) The CRISPR detection system formulation is shown in tables 12 and 13 below:
table 12
TABLE 13
Pre-amplification was performed according to the isothermal amplification method of RT-RAA in example 1, followed by 5
And adding the amplified product of the RT-RAA into a CRISPR detection system, and detecting by adopting a fluorescence method and a test strip method.
(II) detection results
(1) Fluorescence detection result
Fluorescence method: the RAA-CRISPR detection system of the embodiment is placed in a qPCR instrument (the excitation light wavelength is 490 and nm, the emission light wavelength is 520nm, the reaction temperature is 37 ℃, fluorescence is collected once every 1 min for 35 times), the fluorescence intensity change in the detection system is recorded, and the fluorescence signal intensity of an end point is recorded. The fluorescence detection results of the CRISPR-Cas13a detection system are shown in table 14:
TABLE 14
Analysis of results:
(1) Conventional CRISPR detection systems are stable in storage at-20 ℃;
(2) the conventional CRISPR detection system is unstable in storage at 25 ℃ and is not beneficial to the industrialized production of reagents;
(3) in the novel CRISPR detection system (group 1), as shown in group 1, namely, a reaction system of RAA (containing a report probe) and CRISPR (not containing a report probe) can be stably stored for 3 days at 25 ℃, is consistent with the storage lower limit of-20 ℃ and has higher fluorescence value, so that the novel CRISPR detection system is convenient for daily operation and transportation, and meets the freeze-drying requirement.
(2) Test result-line display method by test paper strip method
Test strip method: the RAA-CRISPR detection system of the embodiment is placed in a thermostat for incubation at 37 ℃ for 20min, and then a test strip (purchased from Shanghai Liaorun Biotechnology Co., ltd.) is loaded for detection. The detection results of the CRISPR-Cas13a detection system by the line display method are shown in Table 15:
TABLE 15
Note that: the method is line display detection, T is a detection site, and C is a quality control site. T+ represents a detected report in the product, and the result is positive; t-represents no reporter RNA detected in the product and the result is negative. Negative. C+ represents qualified quality control, and the result is effective; c-represents unqualified quality control, and results are invalid.
Analysis of results:
(1) conventional CRISPR detection systems are stable in storage at-20 ℃;
(2) The conventional CRISPR detection system is unstable in storage at 25 ℃ and is not beneficial to the industrialized production of reagents;
(3) in the novel CRISPR detection system, as shown in a group 1, namely, a reaction system of RAA (containing a reporter probe) and CRISPR (not containing the reporter probe) can be stably stored for 3 days at 25 ℃, is consistent with the storage lower limit of-20 ℃, and is convenient for daily operation and transportation and meets the freeze-drying requirement.
(2) Test result by test paper strip method-line eliminating method
The detection results of the vanishing line method of the CRISPR-Cas13a detection system are shown in Table 16:
table 16
Note that: the method is a line elimination method detection, T is a detection site, and C is a quality control site. T+ represents that the report in the product is not completely degraded by Cas13a, and the result is negative; the T-representative product reported complete degradation by Cas13a, resulting in a positive result. C+ represents qualified quality control, and the result is effective; c-represents unqualified quality control, and results are invalid.
Analysis of results:
(1) conventional CRISPR detection systems are stable in storage at-20 ℃;
(2) the conventional CRISPR detection system is unstable in storage at 25 ℃ and is not beneficial to the industrialized production of reagents;
(3) in the novel CRISPR detection system, as shown in a group 1, namely, a reaction system of RAA (containing a reporter probe) and CRISPR (not containing the reporter probe) can be stably stored for 3 days at 25 ℃, is consistent with the storage lower limit of-20 ℃, and is convenient for daily operation and transportation and meets the freeze-drying requirement.
In conclusion, the report RNA is added into the RT-RAA system from the CRISPR detection system, so that the stability and the detection performance of the RT-RAA+CRISPR detection system can be improved. The detection of low copy samples has a remarkable effect. Meanwhile, the stability of the CRISPR reagent is increased, the preservation time of the CRISPR reagent is prolonged, and the industrial production is facilitated.
Example 4 compatibility of novel CRISPR detection System with other amplification methods
In this example, the Cas13a detection system was taken as an example, and the reporter RNA was adjusted under other isothermal amplification and PCR amplification systems to verify the effect on system stability. In this example, the monkey poxvirus F3L gene SEQ ID NO:1 detection system to detect and verify DNA target and RNA target in sample.
The research method comprises the following steps:
1) A plurality of amplification systems of the simian poxvirus F3L pseudovirus extract products are amplified, and the amplification loading amount is 10copy/T and 1copy/T.
2) The isothermal amplification system adopts LAMP loop-mediated isothermal amplification technology, RCA rolling circle amplification technology, CPA cross primer amplification technology, SDA strand displacement amplification technology and HDA helicase dependent amplification technology respectively.
3) And (3) designing Cas13a gRNA, configuring different amplification systems-CRISPR detection systems, wherein group 1 is a novel CRISPR detection system, the control group, the on-site matched group and the-20 ℃ control group are conventional CRISPR detection systems, and after the group 1 and the control group are stored for 3 days at 25 ℃, detection is carried out, and comparison is carried out with the control group and the on-site matched group at-20 ℃, and the specific is shown in tables 17-19.
TABLE 17
TABLE 18
TABLE 19
Pre-amplification was performed according to the amplification method of example 1, followed by taking 5
And adding the amplified product of L into a CRISPR detection system, and detecting by adopting a fluorescence method and a test strip.
(II) detection results
(1) Fluorescence detection result
The above detection system of this example was placed in a qPCR apparatus (excitation light wavelength 490, nm, emission light wavelength 520, nm, reaction temperature 37 ℃ C.; fluorescence was collected every 1 min for 35 times total), the fluorescence intensity change in the detection system was recorded, and the end point fluorescence signal intensity was recorded, and the detection results were shown in tables 20 to 22.
Table 20
Table 21
Table 22
Analysis of results:
(1) the conventional CRISPR detection system is unstable when stored at 25 ℃ for 3 days, and cannot finish stable amplification of 1copy after being combined with LAMP, RCA, SDA, CPA, HDA, PCR amplification products as shown by group control in tables 20-22, so that the reagent industrialized production is not facilitated;
(2) the novel CRISPR detection system has good compatibility with LAMP, RCA, SDA, CPA, HDA, PCR amplification products, the CRISPR detection can be successfully completed by various amplification method products, the CRISPR detection system is stable at 25 ℃ for 3 days, and can reach 1copy when combined with LAMP, CPA, SDA, CPA, HDA, PCR amplification products and stored at-20 ℃ and the lower limit of the CRISPR detection system configured at present, and the fluorescent value is good, so that the CRISPR detection system is convenient for daily operation and transportation, and meets the freeze-drying requirement.
(2) Test result-line display method by test paper strip method
After the detection system of the embodiment is placed in a constant temperature instrument for incubation at 37 ℃ for 20min, test strips (purchased from Shanghai Liangrun biotechnology Co., ltd.) are loaded and detected, and the detection results of the line display method are shown in tables 23-25.
Table 23
Table 24
Table 25
Note that: the method is line display detection, T is a detection site, and C is a quality control site. T+ represents a detected report in the product, and the result is positive; t-represents no reporter RNA detected in the product and the result is negative. C+ represents qualified quality control, and the result is effective; c-represents unqualified quality control, and results are invalid.
Analysis of results:
(1) the conventional CRISPR detection system is unstable when stored at 25 ℃ for 3 days, and cannot finish stable amplification of 1copy after being combined with LAMP, RCA, SDA, CPA, HDA, PCR amplification products as shown by group control in tables 20-22, so that the reagent industrialized production is not facilitated;
(2) the novel CRISPR detection system has good compatibility with LAMP, RCA, SDA, CPA, HDA, PCR amplification products, the CRISPR detection can be successfully completed by various amplification method products, the CRISPR detection system is stable in storage for 3 days at 25 ℃, is combined with LAMP, CPA, SDA, CPA, HDA, PCR amplification products as shown by the detection results of group 1 in tables 20-22, is consistent with the lower limit of the CRISPR detection system stored at-20 ℃ and configured at present, can reach 1copy, has good fluorescence value, and is convenient for daily operation and transportation, and the freeze-drying requirement is met.
(3) Test result by test paper strip method-line eliminating method
After the detection system of the embodiment is placed in a thermostat for incubation at 37 ℃ for 20min, test strips (purchased from Shanghai Liang run Biotechnology Co., ltd.) are loaded and detected, and the detection results of the line display method are shown in tables 26-28.
Table 26
Table 27
Table 28
Note that: the method is a line elimination method detection, T is a detection site, and C is a quality control site. T+ represents that the report in the product is not completely degraded by Cas13a, and the result is negative; the T-representative product reported complete degradation by Cas13a, resulting in a positive result. C+ represents qualified quality control, and the result is effective; c-represents unqualified quality control, and results are invalid.
Analysis of results:
(1) the conventional CRISPR detection system is unstable when stored at 25 ℃ for 3 days, and cannot finish stable amplification of 1copy after being combined with LAMP, CPA, SDA, CPA, HDA, PCR amplification products as shown by group control in tables 20-22, so that the reagent industrialized production is not facilitated;
(2) the novel CRISPR detection system has good compatibility with LAMP, CPA, SDA, CPA, HDA, PCR amplification products, CRISPR detection can be successfully completed by various amplification method products, the CRISPR detection system is stable when stored at 25 ℃ for 3 days, and can reach 1copy when combined with LAMP, CPA, SDA, CPA, HDA, PCR amplification products and stored at-20 ℃ and the lower limit of the CRISPR detection system which is configured at present as shown in group 1 in table 20-table 22, thus being convenient for daily operation and transportation and meeting the freeze-drying requirement.
The above-described performance analysis of cas13a suggests that the reason for the improvement in stability of CRISPR reagent is that cas13a has a certain RNA cleavage activity even when it is not activated, resulting in a decrease in storage stability of reporter RNA and gRNA, a decrease in activity of cas13a, and an unstable detection limit when it is detected by a fluorescence method and a dipstick method. The report probe is transferred into an amplification system, so that the stability of the detection system can be improved, and the stable detection of the CRISPR detection system is facilitated.
Example 5 Effect of novel CRISPR detection System under other storage conditions
In this example, the Cas13a detection system was taken as an example, and the effect of low-temperature preservation at-20 ℃ was verified by adjusting the reporter RNA or the reporter probe in the RAA isothermal amplification system.
In this example, the fluorescent detection reagents were prepared, and the novel CRISPR detection system, the conventional CRISPR detection system, were stored after being prepared, and were stored at-20℃for detection of 10copy/T and 1copy/T, NTC for 6 months, 12 months, 18 months and 24 months, respectively, and the detection results are shown in Table 29 and FIGS. 1 and 2.
Table 29
The experimental comparison shows that the stability at-20 ℃ is verified. When the novel CRISPR detection system is preserved at the temperature of minus 20 ℃, the detection lower limit is 1copy/T when the preservation time of the novel CRISPR detection system is 24 months; whereas the lower limit of detection of the conventional system is 10copy/T in 18 months of storage, and 10 copies cannot be detected in 24 months when the fluorescence value is remarkably reduced (the lower limit is 1000 copies through experimental verification). The novel CRISPR detection system has better low-temperature storage stability. The novel CRISPR detection system is more beneficial to long-term preservation of the CRISPR detection method.
Example 6 novel lyophilized microsphere of CRISPR detection System
In this example, the reagents of group 1 and the control group in example 2 and the reagents of group 1 and the control group in example 3 were lyophilized according to the following protocol:
and (3) freeze-drying:
1) Lyoprotectant: respectively preparing 20% bovine serum albumin (W/V), 8% threonine (W/V) and 25% PEG20000 (W/V), 4
m filter membrane is used after filtration.
2) And (3) freeze-drying: the freeze-drying protective agent is prepared according to 20 percent bovine serum albumin 3
l/T, 8% threonine 3
l/T、25%PEG20000 4
And mixing the reagent I with the RAA amplification reagent and the CRISPR detection reagent respectively. Dripping by adopting a continuous liquid separator after uniformly mixingPrefreezing in liquid nitrogen.
3) Prefreezing the frozen reagent was transferred to a penicillin bottle and placed in a vacuum desiccator that had been pre-cooled. Vacuum drying at-50deg.C for 10 hr; heating to-30deg.C, vacuum drying for 3 hr; heating to-10deg.C, vacuum drying for 3 hr; heating to 20 ℃ and vacuum drying for 3h.
4) After lyophilization was completed, the vial was closed under vacuum.
The lyophilized pellet was stored at 25 ℃ for 3, 6, 12, 18 months and compared to the results of the new lyophilized reagents.
(1) Fluorescence detection result
The Cas13a freeze-dried sphere fluorescence assay results are shown in table 30:
table 30
The Cas12a freeze-dried sphere fluorescence assay results are shown in table 31:
Table 31
Analysis of results:
(1) the novel RAA and CRISPR detection system has stable performance of storing the Cas12a and Cas13a freeze-dried balls for 18 months, and the detection fluorescence value and the detection lower limit 1copy/T have no obvious difference with the novel freeze-dried reagent;
(2) the conventional CRISPR detection system has poor storage stability of Cas12a in a Cas13a freeze-dried ball, the lower detection limit of 10copy/T is stored for 12 months, the signal value is obviously reduced, and 10 copies cannot be detected (the lower detection limit of 500copy/T is verified) for 18 months;
(2) Test result-line display method by test paper strip method
The Cas13a freeze-dried sphere visualization assay results are shown in table 32:
table 32
The Cas12a freeze-dried sphere visualization assay results are shown in table 33:
table 33
Note that: the method is line display detection, T is a detection site, and C is a quality control site. T+ represents that the product detects the report probe, and the result is positive; t-represents no reporter probe detected in the product and the result is negative. Negative. C+ represents qualified quality control, and the result is effective; c-represents unqualified quality control, and results are invalid.
Analysis of results:
(1) the novel RAA and CRISPR detection system has stable performance of storing the Cas12a and Cas13a freeze-dried balls for 18 months, and the detection lower limit of 1copy/T has no obvious difference with the novel freeze-dried reagent;
(2) the conventional CRISPR detection system has poor storage stability of Cas12a in a Cas13a freeze-dried ball, and cannot be detected at a lower detection limit of 10 copies/t (a lower detection limit of 500 copies/t for verification) for 12 months and cannot be detected at a lower detection limit of 1000 copies/t for 18 months;
(3) Test result by test paper strip method-line eliminating method
The Cas13a freeze-dried ball vanishing line detection results are shown in table 34:
watch 34
The Cas12a freeze-dried ball vanishing line detection results are shown in table 35:
table 35
Note that: the method is a line elimination method detection, T is a detection site, and C is a quality control site. T+ represents that the product detects the report probe, and the result is positive; t-represents no reporter probe detected in the product and the result is negative. C+ represents qualified quality control, and the result is effective; c-represents unqualified quality control, and results are invalid.
Analysis of results:
(1) the novel RAA and CRISPR detection system has stable performance of storing the Cas12a and Cas13a freeze-dried balls for 18 months, and the detection lower limit of 1copy/T has no obvious difference with the novel freeze-dried reagent;
(2) the conventional CRISPR detection system has poor storage stability of the Cas12a in the freeze-dried spheres of the Cas13a, wherein the lower detection limit of 10copy/t of 12 months of storage of the Cas13a is not detected (the lower detection limit of 50copy/t of the verified detection) and the lower detection limit of 10copy/t of 12 months of storage of the Cas12a is not detected. The copy could not be detected (lower limit of 1000copy/t for the verified detection) after 18 months of 10 copies;
in conclusion, the report RNA/probe is added into the RAA system from the CRISPR detection system, so that the stability and detection performance of the CRISPR freeze-dried sphere detection system can be improved, and the stability after 12 months is more remarkable. The novel scheme can remarkably improve the long-term stability of the freeze-dried balls and can meet the industrialization demand.
Example 7 Effect of novel CRISPR detection System on CrRNA screening
In this example, the Cas13a detection system is taken as an example, and the effect of the report RNA on the design requirement of crRNA is verified under the RAA isothermal amplification system.
In this example, according to the crRNA design principle, 50 different crrnas are continuously designed to synthesize by adopting the design principle of a general sequence of +28bp in the repeated sequence, as shown in table 36. The RAA+CRISPR reaction was performed after synthesis.
Table 36
The same RAA products (positive and negative) are detected by using a CRISPR detection system, and are interpreted according to the endpoint fluorescence value. Interpretation criteria: the difference between the fluorescence values of the positive sample and the negative sample is more than 5 times, so that the distinction is considered to be obvious, and crRNA can be used; if the discrimination is not significant, it indicates that crRNA cannot be used. The statistics of the available results for the 50 crrnas described above are shown in table 37 below.
Table 37
Experimental comparison shows that the novel CRISPR detection system is adopted, and the number of available CRRNAs in the same CRRNA library is obviously increased compared with that of the conventional CRISPR detection system. It is demonstrated that in the novel CRISPR detection system, the screening requirements for guide RNAs are less stringent than in conventional CRISPR detection systems, and the construction of nucleic acid amplification-CRISPR detection systems is easier to accomplish.
Example 8 Effect of novel CRISPR detection System on CRISPR raw Material use
The embodiment verifies that the novel CRISPR detection system can reduce the cost on the premise of not influencing the detection performance by reducing the detection usage amount of the Cas effect protein.
Study method:
1) The control group was formulated as in example 2 group 1 (Cas 12a, cas12 b) and example 3 group 1 (Cas 13 a).
2) The experimental groups reduced the cas protein dose by 10% (22.5 nM), 20% (20 nM), 30% (17.5 nM) and the remaining dose was unchanged.
3) The detection templates are respectively 10 copy/T3 compound holes, 1 copy/T3 compound holes and 3 compound holes of purified water.
4) The system is tested after being placed for 3 days at 25 ℃ after the preparation, and the testing performance is verified by a fluorescence method.
Verification systems for Cas12a, cas12b, cas13a are formulated as shown in table 38 below.
Table 38
(II) detection result:
(1) Fluorescence detection result
The test results of cas12a assay are shown in Table 39 below:
table 39
The test results of cas12b are shown in Table 40:
table 40
The test results of cas13a are shown in Table 41:
table 41
Analysis of results:
novel CRISPR detection system, cas12a, cas12b and cas13a are used in low amounts of 20nM, and the lower limit of 3 days detection is as follows: 1copy/T, and stable performance.
After the dosage of cas12a, cas12b and cas13a is reduced in the conventional CRISPR detection system, the detection performance is reduced after the detection system is stored for 3 days at 25 ℃, wherein: the lower detection limit is 10copy/T when the dosages of cas12a, cas12b and cas13a are 25nM, 22.5nM and 20 nM; at 17.5nM, 10copy/T was undetectable (verified limit of detection was 1000 copy/T).
(2) Test result-line display method by test paper strip method
The test results of cas12a are shown in Table 42:
table 42
The test results of cas12b are shown in Table 43:
table 43
The test results of cas13a are shown in Table 44:
table 44
Analysis of results:
novel CRISPR detection system, cas12a, cas12b and cas13a are used in low amounts of 20nM, and the lower limit of 3 days detection is as follows: 1copy/T, and stable performance.
After the dosage of cas12a, cas12b and cas13a is reduced in the conventional CRISPR detection system, the detection performance is reduced after the detection system is stored for 3 days at 25 ℃, wherein: the lower detection limit is 10copy/T when the dosage of cas12a, cas12b and cas13a is 25nM and 22.5 nM; at 20nM and 17.5nM, 10copy/T was undetectable (verified limit of detection was 1000 copy/T).
(3) Test result by test paper strip method-line eliminating method
The test results of cas12a are shown in Table 45:
table 45
The test results of cas12b assay are shown in Table 46:
Watch 46
The test results of cas13a are shown in Table 47:
table 47
Analysis of results:
novel CRISPR detection system, cas12a, cas12b and cas13a are used in low amounts of 20nM, and the lower limit of 3 days detection is as follows: 1copy/T, and stable performance.
After the dosage of cas12a, cas12b and cas13a is reduced, the conventional CRISPR detection system is preserved for 3 days at 25 ℃, and after the dosage of cas12a, cas12b and cas13a is reduced, the detection performance is reduced after the conventional CRISPR detection system is preserved for 3 days at 25 ℃, wherein: the lower detection limit is 10copy/T when the dosage of cas12a, cas12b and cas13a is 25 nM; at 22.5nM, 20nM, 17.5nM, 10copy/T was undetectable (verified limit of detection is 1000 copy/T).
In summary, the report RNA/probe is added into the RPA system from the CRISPR detection system, so that the dosage of cas enzyme by the CRISPR detection system can be reduced, the dosage of cas enzyme by 20% is reduced, the detection performance and the 3-day stability of the CRISPR detection reagent are not obviously affected, the dosage of the reagent can be reduced on the basis of ensuring the detection performance, and the cost is reduced.
The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The foregoing examples represent only a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application is to be determined by the claims appended hereto.
Claims (29)
1. A nucleic acid detection system comprising a CRISPR detection system comprising a Cas effect protein and no nucleic acid signaling molecule and a nucleic acid amplification system comprising a nucleic acid signaling molecule, the CRISPR detection system and the nucleic acid amplification system being combined to detect a target nucleic acid, the nucleic acid signaling molecule being configured to report a detection signal.
2. The nucleic acid detection system of claim 1, wherein the CRISPR detection system further comprises a guide RNA for binding to a corresponding target nucleic acid.
3. The nucleic acid detection system of claim 1, wherein the nucleic acid amplification system further comprises a guide RNA for binding to a corresponding target nucleic acid.
4. The nucleic acid detection system of claim 2 or 3, wherein the target nucleic acid is a target RNA and the CRISPR detection system further comprises an RNA polymerase.
5. The nucleic acid detection system of claim 4, wherein the RNA polymerase comprises at least one of T7 RNA polymerase and Sp6 RNA polymerase.
6. The nucleic acid detection system of claim 4, wherein the nucleic acid amplification system further comprises amplification primers with an RNA polymerase recognition site.
7. The nucleic acid detection system of claim 4, wherein the Cas effector protein is an RNA targeting effector protein.
8. The nucleic acid detection system of claim 7, wherein the RNA targeting effector protein is Cas13a and/or Cas13b.
9. The nucleic acid detection system of claim 4, wherein the nucleic acid signal molecule comprises RNA and a detection signal is generated based on cleavage of the RNA.
10. The nucleic acid detection system of claim 4, wherein the CRISPR detection system further comprises rtps, rnase inhibitors, mg 2+ And HEPES.
11. The nucleic acid detection system of claim 10, wherein the working concentration of each component in the CRISPR detection system satisfies at least one of the following features (1) - (8):
(1) Working concentration of gRNA is 5nM-50
M；
(2) The working concentration of the Cas effect protein is 5nM-500nM;
(3) The working concentration of RNA polymerase is 0.5U-10U;
(4) The working concentration of rNTPs is 1mM-8mM;
(5) The working concentration of the nucleic acid signal molecules is 1nM-2000nM;
(6) The working concentration of the RNase inhibitor is 0.5U-10U;
（7）Mg 2+ the working concentration of (2) is 5mM-20mM;
(8) HEPES works at a concentration of 5mM-20mM.
12. The nucleic acid detection system of claim 2 or 3, wherein the target nucleic acid is a target DNA.
13. The nucleic acid detection system of claim 12, wherein the Cas effector protein is a DNA targeting effector protein.
14. The nucleic acid detection system of claim 13, wherein the DNA targeting effector protein comprises Cas12 and/or Cas14, the Cas12 comprising at least one of Cas12a, cas12b, cas12 c.
15. The nucleic acid detection system of claim 12, wherein the nucleic acid signal molecule comprises DNA, and wherein the detection signal is generated based on cleavage of the DNA.
16. The nucleic acid detection system of claim 12, wherein the CRISPR detection system further comprises dNTPs, mg 2+ At least one of HEPES and gRNA.
17. The nucleic acid detection system of claim 16, wherein the working concentration of each component in the CRISPR detection system satisfies at least one of the following features (1) - (6):
(1) Working concentration of gRNA is 5nM-50
M；
(2) The working concentration of the Cas effect protein is 5nM-500nM;
(3) The working concentration of the nucleic acid signal molecules is 1nM-2000nM;
(4) The working concentration of Mg2+ is 5mM-20mM;
(5) HEPES works at a concentration of 5mM-20mM;
(6) The working concentration of each base in dNTPs is 1mM-8mM.
18. The nucleic acid detection system of claim 1, wherein the nucleic acid amplification system is configured to perform any one of the following amplification methods:
recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, rolling circle amplification, cross primer isothermal amplification, strand displacement amplification, helicase dependent amplification, and PCR amplification.
19. The nucleic acid detection system of claim 2 or 3, wherein the nucleic acid amplification system and/or CRISPR detection system further comprises a lyoprotectant.
20. A nucleic acid diagnostic device comprising one or more individual modules, each individual module comprising the nucleic acid detection system of any one of claims 1 to 19.
21. The nucleic acid diagnostic device of claim 20, wherein the stand-alone module comprises one or more containment units, each containment unit being provided with a nucleic acid amplification system and/or a CRISPR detection system in the nucleic acid detection system.
22. The nucleic acid diagnostic device as claimed in claim 21, wherein the containing unit is for containing at least one of a liquid and a solid, preferably a solid.
23. The nucleic acid diagnostic device of claim 22, wherein the reagents contained in the nucleic acid amplification system and/or the CRISPR detection system are solid.
24. The nucleic acid diagnostic device of claim 22, wherein the solid comprises any one of lyophilized microspheres, lyophilized cake, lyophilized powder, and spots depending on the presence of a solid medium.
25. A method for detecting a target nucleic acid in a sample, characterized in that the sample is contacted with the nucleic acid detection system of any one of claims 1 to 19 for reaction to determine the target nucleic acid in the sample.
26. A method for detecting a target nucleic acid in a sample, comprising the steps of:
adding a sample or a sample set to the nucleic acid diagnostic device of any one of claims 20-24, and contacting the nucleic acid amplification system and the CRISPR detection system in the containment unit to generate a detectable signal;
Detecting the detectable signal, and determining one or more target nucleic acids in the sample.
27. The method of claim 26, wherein the nucleic acid amplification system is configured to perform any one of the following methods:
recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, rolling circle amplification, cross primer isothermal amplification, strand displacement amplification, helicase dependent amplification, and PCR amplification.
28. The method of any one of claims 25 to 27, wherein the target nucleic acid in the sample is determined by fluorescence or lateral flow immunochromatography;
optionally, the lateral flow immunochromatography performs at least one of a line display method and a line elimination method.
29. A method for preparing a nucleic acid detection kit, comprising:
the nucleic acid amplification system and the CRISPR detection system in the nucleic acid detection system according to any one of claims 1 to 19, respectively mixing with a lyoprotectant to obtain a mixture, and subjecting the mixture to lyophilization.
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