CN117737208A - Composition and kit for detecting RNA at room temperature - Google Patents
Composition and kit for detecting RNA at room temperature Download PDFInfo
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Abstract
The invention discloses a composition and a kit for detecting RNA at room temperature, wherein the composition comprises the following components: reverse transcription primer and reverse transcriptase of target RNA, which are used for synthesizing reverse complementary sequence of target RNA, the length of reverse transcription primer is 10-20 nucleotides; the method comprises the steps of (1) an RPA reverse primer, an RPA forward primer, a recombinase, a DNA polymerase and a T7RNA polymerase, wherein the RPA reverse primer, the recombinase, the DNA polymerase and the T7RNA polymerase are used for amplifying a double-stranded DNA amplicon of target RNA, and synthesizing long-chain RNA containing a reverse complementary sequence of the target RNA by taking the double-stranded DNA amplicon as a substrate; the double-stranded DNA amplicon is formed by base complementary pairing of target RNA and a reverse complementary sequence thereof, and the 5' -end of the RPA reverse primer comprises a T7RNA polymerase promoter sequence; cas13a protein and crRNA thereof, crRNA is used for targeted recognition of reverse complementary sequence of target RNA, cas13a protein is used for cleavage of reverse complementary sequence of target RNA; taqMan fluorescent probes, which can be nonspecifically cleaved by Cas13a protein when the reverse complement of the target RNA is cleaved, release the fluorescent group. The composition can detect 0.5 cp/. Mu.L of target RNA at 20 ℃ within 30 min.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a composition and a kit for detecting RNA at room temperature.
Background
Currently, widely used pathogen detection methods are antigen detection and quantitative polymerase chain reaction (qPCR). qPCR technology is very sensitive, but has long reaction time, depends on professional operation technology and expensive equipment, and is not suitable for instant detection. Antigen detection can produce results in 15-30 minutes at room temperature, however, its sensitivity is significantly lower than qPCR, resulting in low accuracy of results and high false negative rate. Isothermal amplification methods, such as Recombinase Polymerase Amplification (RPA) and loop-mediated isothermal amplification (LAMP), are rapid in reaction speed, can produce results within 5-30 minutes, and have high sensitivity similar to qPCR, but poor specificity and false positive rate.
CRISPR-Cas (CRISPR-associated protein) systems have now been used to develop pathogen detection kits. Cas12 or Cas13, after specific recognition of the guide RNA (gRNA) and cleavage of the target nucleic acid sequence, has its non-specific nuclease activity activated, rapidly cleaving single-stranded RNA or single-stranded DNA, a phenomenon known as trans-cleavage. A fluorescent group and a fluorescence quenching group are respectively marked at two ends of single-stranded DNA or single-stranded RNA, and the fluorescent reporter gene is prepared by the fluorescent groups, and the Cas protein can cut the reporter gene to generate an obvious fluorescent signal which can be detected by a fluorescence reader. The CRISPR-Cas has high specificity and high reaction speed, and results are obtained within 5-10 minutes, so that the CRISPR-Cas has excellent application potential in the aspect of nucleic acid detection. However, CRISPR-Cas has low sensitivity, and Cas13 nuclease alone requires several hours to reach attomole level sensitivity, so combined isothermal amplification and CRISPR are the best solution for diagnosing viral samples. The method of Charlock (SHERLOCK) based on Cas13a is divided into two steps of RPA amplification and CRISPR detection, and the sensitivity is obviously improved compared with that of Cas 13. However, integration of multiple steps increases complexity, extends reaction time, increases risk of cross-contamination, and the like. Integrating isothermal amplification and CRISPR detection in one step can reduce the complexity and risk of cross-contamination of a two-step method, a technique known as a one-step nucleic acid detection method. Classical one-step SHINE based on Cas13a achieves pathogen detection by combining recombinase polymerase isothermal amplification technology and LwaCas13a cleavage technology. At 37 ℃, the SHINE starts reverse transcription process by using 30 nucleotide RPA reverse primer, synthesizes cDNA of pathogen RNA, amplifies double-stranded DNA amplicon by using RPA forward primer containing T7RNA polymerase promoter sequence at 5' end, T7RNA polymerase recognizes promoter sequence on amplicon, and transcribes short-chain RNA identical to pathogen RNA sequence, then LwaCas13a recognizes and cleaves specific region of short-chain RNA under crRNA guidance, nonspecific cleavage activity is activated, and rapidly cleaves RNA reporter molecule, releasing fluorescent group. The fluorescent signal read by the fluorescent reader is a positive result of pathogen detection, and if the fluorescent signal is not present, the result is a negative result. Most CRISPR detection methods have a sensitivity far exceeding that of antigen testing, but they require the purchase of temperature control equipment, are costly, and limit their use in point-of-care detection. Therefore, the isothermal amplification and the reaction activity of the Cas13a in one step at room temperature are improved, so that the detection rate and sensitivity are equal to those of the Cas13a at 37 ℃, a temperature control device is eliminated, the cost is reduced, and the Cas13a is more suitable for being developed into a rapid and instant nucleic acid detection method and is more suitable for self-detection and bedside diagnosis.
Disclosure of Invention
Aiming at the problems of long reaction time, low sensitivity, high cost and the like of the existing Cas13a detection method, the reverse complement sequence of the target RNA is used as a cutting target of Cas13a, so that interference of Cas13a cutting on reverse transcription and isothermal amplification reaction is reduced; meanwhile, the length of the reverse transcription primer is shortened, the reaction rate is accelerated, the concentrations of the RPA reverse primer, the RPA forward primer, the reverse transcription primer and the TaqMan fluorescent probe are optimized, the temperature range and the detection sensitivity of the reaction are widened, and the capability of detecting 0.5 cp/. Mu.L SARS-CoV-2RNA within 30 minutes at 20 ℃ is realized.
The technical scheme provided by the invention is as follows:
in a first aspect, the invention provides a composition for room temperature detection of RNA comprising:
reverse transcription primer and reverse transcriptase of target RNA, which are used for synthesizing reverse complementary sequence of target RNA, the length of the reverse transcription primer is 10-20 nucleotides;
the method comprises the steps of (1) an RPA reverse primer, an RPA forward primer, a recombinase, a DNA polymerase and a T7RNA polymerase, wherein the RPA reverse primer, the recombinase, the DNA polymerase and the T7RNA polymerase are used for amplifying a double-stranded DNA amplicon of target RNA, and synthesizing long-chain RNA containing a reverse complementary sequence of the target RNA by taking the double-stranded DNA amplicon as a substrate; the double-stranded DNA amplicon is formed by base complementary pairing of target RNA and a reverse complementary sequence thereof, and the 5' end of the RPA reverse primer comprises a T7RNA polymerase promoter sequence;
a Cas13a protein and crrnas thereof, the crrnas being for targeted recognition of a reverse complement of a target RNA, the Cas13a protein being for cleavage of the reverse complement of the target RNA;
TaqMan fluorescent probes, which can be nonspecifically cleaved by Cas13a protein when the reverse complement of the target RNA is cleaved, release the fluorescent group.
In some embodiments of the invention, the reverse transcription primer is 12 to 16 nucleotides in length, and exemplary reverse transcription primers are 12, 13, 14, 15, or 16 nucleotides in length.
In some preferred embodiments provided herein, the reverse transcription primer is 14 nucleotides in length.
In some embodiments provided herein, the Cas13a protein is at least one of lwcas 13a, lbcas 13a, lshCas13a, pprCas13a, ereCas13a, lneCa3a, camCas13a, rcaCas13a, hhcas 13a, lbuCas13a, lseCas13a, lbmCas13a, lbnncas 13a, rccas 13a, rccdcas 13a, cgCas13a, cg2Cas13a, lwcas 13a, lbfCas13a, lba4Cas13a, lba9Cas13a, lneCas13a, hhcas 13a, rcaCas13 a.
In some preferred embodiments provided herein, the Cas13a protein is LwaCas13a or LbuCas13a.
In some embodiments of the invention provided herein, the concentration of reverse transcription primer of the target RNA is 100-1000nM; the concentration of the RPA reverse primer is 50-500nM; the concentration of the RPA forward primer is 150-1000nM.
In some embodiments provided herein, the concentration of TaqMan fluorescent probes is 100-1000nM.
In some embodiments provided herein, the fluorescence quenching group of the TaqMan fluorescent probe is BHQ, BHQ1, BHQ2 or TAMRA, and the fluorescence reporting group is FAM, CY3, CY5, HEX or TET.
In some embodiments of the invention, the target RNA is SARS-CoV-2RNA, which has the sequence ACUGAGAUCUUUCAUUUUACCGUCACCA (see SEQ ID NO: 2);
the crRNA is
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUGGUGACGGU AAAAUGAAAGAUCUCAGU (see SEQ ID NO: 10);
the sequence of the reverse transcription primer of the target RNA is as follows: TTTTGGTGTATTCA (see SEQ ID NO: 17); the sequence of the RPA reverse primer is:
CCGGTAATACGACTCACTATAGGGCCCATATGATGCCGTCTTTG (see SEQ ID NO: 16);
the sequence of the RPA forward primer is: CCTCGAGGACAAGGCGTTCCAATTAA (see SEQ ID NO: 15);
the RNA sequence of the TaqMan fluorescent probe is at least one of UUUUU, UUUUUU, UUUUUUU, UUUUUUUU.
In some preferred embodiments provided by the invention, the TaqMan fluorescent probe is FAM-UUUU-BHQ 1.
In some embodiments of the invention, the room temperature nucleic acid detection kit further comprises an RNase inhibitor, an RNase H, ribonucleoside triphosphates, deoxyribonucleoside triphosphates, a buffer, and a reaction activator.
In a second aspect, the invention provides a room temperature nucleic acid detection kit comprising a composition for room temperature detection of RNA as described above.
In some embodiments provided herein, the room temperature nucleic acid detection kit comprises: 20-200nM LwaCas13a or LbuCas13a,10-100nM crRNA, 1.5-2.5U/. Mu.L reverse transcriptase, 50-500 ng/. Mu.L T7RNA polymerase, 100-1000nM TaqMan fluorescent probe, 50-500nM RPA reverse primer, 150-1000nM RPA forward primer, 100-1000nM reverse transcription primer.
In some preferred embodiments provided herein, the room temperature nucleic acid detection kit further comprises: 1U/. Mu.L of RNase inhibitor, 0.05U/. Mu.L of RNase H,2mM each of the four rNTPs.
Compared with the prior art, the invention has the following beneficial effects:
the composition for detecting RNA at room temperature provided by the invention can rapidly and sensitively detect target RNA at 20-37 ℃ and can realize the capability of detecting 0.5 cp/. Mu.L target RNA at 20 ℃ for 30 minutes.
Drawings
FIG. 1.REVERSE one-step method is faster and more sensitive than classical SHINE method. (a) Schematic of reverse transcription, recombinant polymerase isothermal amplification RPA, in Vitro Transcription (IVT) and CRISPR mediated detection processes. (b) comparing amplicon accumulation in REVERSE and SHINE. RPA reactions and LwaCas13a/crRNA were incubated at 37 ℃ for 0,5, 10, 15, 20, 25, 30 minutes. The resulting amplicon was analyzed by TAE gel electrophoresis. Arrows indicate amplicon bands. (c) testing for interference of lwaca 13a cleavage with the reverse transcription process. The Orf1ab IVT RNA, reverse transcriptase and Cas13a/crRNA were incubated at 37 ℃ for 5 minutes, and then the cDNA generated was quantified using qPCR. Each group of cDNA doses was normalized to the cDNA level of the "crRNA free" group. (d) comparing fluorescence curves of REVERSE and SHINE. (e-f) comparing sensitivity of REVERSE and SHINE. After incubation at 42 ℃ for 30 minutes, the fluorescence value of the one-step method was determined. Mean ± standard deviation of c-e, n=3-6 technical replicates. (g) REVERSE detects the fluorescence value of SARS-CoV-2 clinical sample. The reaction temperature was 42℃and determined using SpectraMax i3x, the black dashed line represents the threshold value, three times the negative control fluorescence values in e and f.
FIG. 2. One-step method REVERSE-2 at room temperature. (a) testing REVERSE at different temperatures. Fluorescent signals at 25, 35, 45 and 55 minutes were detected using a simple blue light device at 23 ℃,25 ℃,30 ℃ and 37 ℃. The substrate was the SARS-CoV-2N gene RNA of the pseudovirus at 3 cp/. Mu.L extracted. (b-d) the response capacity of REVERSE at 20℃was optimized by increasing the primer dose (b) and the fluorescent reporter gene concentration (c). (b) Reaction rates were tested for 1X, 2X, 4X and 6X primer set concentrations using the 1X ssRNA reporter. (c) The reaction rates of 1X, 2X, 3X and 4X fluorescent reporter concentrations were tested using 1X primer doses. (d) optimized REVERSE. b-d, the reaction substrate is the SARS-CoV-2N gene RNA of the pseudovirus of 4 cp/. Mu.L extracted. (e-f) reading the reaction signal of REVERSE-2 at 20℃using a microplate reader (e) and a blue light device (f). The black dashed line represents a threshold value that is three times the negative control fluorescence value. (g-k) REVERSE-2 detection 107 SARS-CoV-2 nasopharyngeal swab samples, comprising 87 SARS-CoV-2 positive samples (63 extracted RNA and 24 unextracted samples) (g, i), and 20 SARS-CoV-2RNA negative samples (h, j). After reacting for 30 minutes at 20 ℃, blue light images are shot by using a smart phone, and fluorescent values are read by using an enzyme label instrument. The black dashed line indicates the threshold value, which is three times the negative control fluorescence value in g and h. Samples with RT-qPCR Ct values exceeding 38 were classified as negative samples. (k) statistics of results of RT-qPCR and REVERSE-2.
Figure 3 compares the reaction rates of different Cas protein-mediated one-step methods at room temperature. The Cas13a-, cas12a-, and Cas12 b-mediated one-step reactions were compared at 20 ℃ and 25 ℃. The reaction substrate is extracted 1 cp/. Mu.L pseudovirus-SARS-CoV-2N gene RNA. Results were read using a blue light device at 30, 40, 50 and 60 minutes, respectively.
FIG. 4. Sensitivity and specificity of REVERSE-2 and antigen tests were compared at room temperature. (a-b) evaluating sensitivity of REVERSE-2 (a) and antigen test (b). (c-d) testing the specificity of REVERSE-2 and antigen responses.
Fig. 5 is a schematic diagram of the reverse detection method.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments.
Heretofore, the classical one-step approach based on recombinase polymerase amplification with Cas13a was SHINE. The method synthesizes cDNA of pathogen RNA by utilizing RPA reverse primer with 30 nucleotides at 37 ℃, utilizes RPA forward primer with 5' end containing T7RNA polymerase promoter sequence to amplify double-stranded DNA amplicon, then T7RNA polymerase recognizes the promoter sequence on the amplicon, and transcribes short-chain RNA identical with pathogen RNA sequence, then LwaCas13a recognizes and cleaves specific region of short-chain RNA under the guidance of crRNA, nonspecific cleavage activity is activated, RNA reporter molecule is rapidly cleaved, fluorescent group is released, and fluorescent reader reads out result. The sensitivity of SHINE in 40-50 minutes for diagnosing SARS-CoV-2 positive nasopharyngeal swab sample is 100-1000 cp/. Mu.L, and the method has low detection sensitivity and long reaction time, and can not meet the requirement of instant and rapid pathogen nucleic acid detection method.
The present invention discovers that Cas13a directly degrades viral RNA in a SHINE one-step method, interfering with amplification of target sequences, resulting in reduced sensitivity and speed of one-step assays. Based on the above, the invention provides a one-step method capable of avoiding the degradation of viral RNA by Cas13a, and optimizing the dosages of the primer and the RNA reporter molecule, so that the SARS-CoV-2 sample can be detected within 30 minutes at room temperature (20 ℃) with the sensitivity of 0.5 cp/. Mu.L, the efficiency of detecting the viral RNA is obviously improved, and the sensitivity level is comparable with that of RT-qPCR.
In order to develop a rapid detection method based on CRISPR without a temperature control device, the method is superior to the widely used antigen detection method in self-test application. According to the invention, the target RNA sequence of the Cas13a protein is redesigned, the reverse complementary sequence of the target RNA is used as a cutting target of the Cas13a, and the Cas13a is ensured to target only the reverse complementary sequence of the target RNA, but not the target RNA, so that the interference of the cutting of the Cas13a on reverse transcription and isothermal amplification reaction is eliminated, the detection sensitivity is further improved, and the reaction rate is accelerated. Meanwhile, the length of the reverse transcription primer is shortened, the reaction rate is accelerated, in addition, the temperature range and the detection sensitivity of the reaction are widened by optimizing the concentrations of the RPA reverse primer, the RPA forward primer, the reverse transcription primer and the TaqMan fluorescent probe, specifically, the concentrations of the 2-4 times of the reverse transcription primer, the 2-4 times of the concentrations of the RPA reverse primer, the RPA forward primer and the 3-4 times of the concentrations of the TaqMan fluorescent probe are adopted, the temperature range of the one-step detection reaction is enlarged, the pathogen nucleic acid can be rapidly and sensitively detected under the condition of room temperature, and the capability of detecting 0.5 cp/mu L target RNA within 30 minutes at 20 ℃ is finally realized.
According to the above aspects, the present invention provides a composition for detecting RNA at room temperature, comprising:
reverse transcription primer and reverse transcriptase of target RNA, which are used for synthesizing reverse complementary sequence of target RNA, the length of the reverse transcription primer is 10-20 nucleotides;
the method comprises the steps of (1) an RPA reverse primer, an RPA forward primer, a recombinase, a DNA polymerase and a T7RNA polymerase, wherein the RPA reverse primer, the recombinase, the DNA polymerase and the T7RNA polymerase are used for amplifying a double-stranded DNA amplicon of target RNA, and synthesizing long-chain RNA containing a reverse complementary sequence of the target RNA by taking the double-stranded DNA amplicon as a substrate; the double-stranded DNA amplicon is formed by base complementary pairing of target RNA and a reverse complementary sequence thereof, and the 5' end of the RPA reverse primer comprises a T7RNA polymerase promoter sequence;
a Cas13a protein and crrnas thereof, the crrnas being for targeted recognition of a reverse complement of a target RNA, the Cas13a protein being for cleavage of the reverse complement of the target RNA;
TaqMan fluorescent probes, which can be nonspecifically cleaved by Cas13a protein when the reverse complement of the target RNA is cleaved, release the fluorescent group.
In some embodiments of the invention, the reverse transcription primer is 12 to 16 nucleotides in length, and exemplary reverse transcription primers are 12, 13, 14, 15, or 16 nucleotides in length.
In some preferred embodiments provided herein, the reverse transcription primer is 14 nucleotides in length.
In some embodiments provided herein, the Cas13a protein is at least one of lwcas 13a, lbcas 13a, lshCas13a, pprCas13a, ereCas13a, lneCa3a, camCas13a, rcaCas13a, hhcas 13a, lbuCas13a, lseCas13a, lbmCas13a, lbnncas 13a, rccas 13a, rccdcas 13a, cgCas13a, cg2Cas13a, lwcas 13a, lbfCas13a, lba4Cas13a, lba9Cas13a, lneCas13a, hhcas 13a, rcaCas13 a.
In some preferred embodiments provided herein, the Cas13a protein is LwaCas13a or LbuCas13a.
In some embodiments of the invention provided herein, the concentration of reverse transcription primer of the target RNA is 100-1000nM; the concentration of the RPA reverse primer is 50-500nM; the concentration of the RPA forward primer is 150-1000nM.
In some embodiments provided herein, the concentration of TaqMan fluorescent probes is 100-1000nM.
In some embodiments provided herein, the fluorescence quenching group of the TaqMan fluorescent probe is BHQ, BHQ1, BHQ2 or TAMRA, and the fluorescence reporting group is FAM, CY3, CY5, HEX or TET.
In some embodiments of the invention, the target RNA is SARS-CoV-2RNA, which has the sequence ACUGAGAUCUUUCAUUUUACCGUCACCA;
the crRNA is
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUGGUGACGGU AAAAUGAAAGAUCUCAGU；
The sequence of the reverse transcription primer of the target RNA is as follows: TTTTGGTGTATTCA;
the sequence of the RPA reverse primer is:
CCGGTAATACGACTCACTATAGGGCCCATATGATGCCGTCTTTG；
the sequence of the RPA forward primer is: CCTCGAGGACAAGGCGTTCCAATTAA;
the RNA sequence of the TaqMan fluorescent probe is at least one of UUUUU, UUUUUU, UUUUUUU, UUUUUUUU.
In some preferred embodiments provided by the invention, the TaqMan fluorescent probe is FAM-UUUU-BHQ 1.
In some embodiments of the invention, the room temperature nucleic acid detection kit further comprises an RNase inhibitor, an RNase H, ribonucleoside triphosphates, deoxyribonucleoside triphosphates, a buffer, and a reaction activator.
The invention also provides a room temperature nucleic acid detection kit, which comprises the composition for detecting RNA at room temperature.
In some embodiments provided herein, the room temperature nucleic acid detection kit comprises: 20-200nM LwaCas13a or LbuCas13a,10-100nM crRNA, 1.5-2.5U/. Mu.L reverse transcriptase, 50-500 ng/. Mu.L T7RNA polymerase, 100-1000nM TaqMan fluorescent probe, 50-500nM RPA reverse primer, 150-1000nM RPA forward primer, 100-1000nM reverse transcription primer.
In some preferred embodiments provided herein, the room temperature nucleic acid detection kit further comprises: 1U/. Mu.L of RNase inhibitor, 0.05U/. Mu.L of RNase H,2mM each of the four rNTPs.
In the examples provided by the invention, SARS-CoV-2 is taken as pathogen to develop a composition and a kit for detecting RNA at room temperature, the specifically adopted Cas13a is LwaCas13a, and the specifically adopted gene sequences are shown in tables 1-4:
TABLE 1 target RNA
TABLE 2 PCR primers, qPCR primers and RT primers
TABLE 3 crRNA sequence of LwaCas13a
TABLE 4 RPA and RT-RPA primers
The following describes the technical scheme of the present invention in detail through specific embodiments: examples
Development of CRISPR detection method
The present invention notes that the RNA transcribed in vitro in the SHINE method is identical to the RNA sequence of the pathogen to be detected (FIG. 1 a). The present invention speculates that Cas13a may rapidly recognize and cleave viral RNA during the initial stages of the reaction, not only reducing the copy number of pathogen RNA samples, but also interfering with subsequent reverse transcription and isothermal amplification processes. Experiments in the present invention demonstrate that cleavage of Cas13a severely reduces reverse transcription and RPA amplification efficiency (fig. 1 b-c). In addition, it was noted that the SHINE method synthesizes cDNA using 30 nucleotide RPA reverse primer, and that an excessive length of 30 nucleotides reduces the efficiency of reverse transcription and reduces cDNA yield (FIG. 1 a). Therefore, in order to reduce the interference of Cas13a on REVERSE transcription and amplification, increase the efficiency of REVERSE transcription, while retaining the high cleavage activity of Cas13a, the present invention developed a new method (REVERSE), the schematic diagram of which is shown in fig. 5. In REVERSE, the REVERSE complementary sequence cDNA of target RNA is synthesized by using a short REVERSE transcription primer of 14 nucleotides to improve the cDNA synthesis efficiency (figure 1 a). Subsequently, the present invention uses reverse primer containing T7RNA polymerase promoter sequence at 5' end for RPA amplification, this arrangement ensures that RNA transcribed with DNA amplicon as template is reverse complementary to the sequence of viral RNA, allowing Cas13a to specifically cleave only transcribed RNA, avoiding cleavage of viral RNA, ensuring reverse transcription and amplification efficiency (FIG. 1 a). In support of this, in REVERSE, amplicons appeared within 5 minutes, a large amount of amplicons accumulated in 10 minutes and accumulated over time, whereas amplicons appeared only in 10 minutes in she, the amplicon dose was always low even if the reaction time was prolonged (fig. 1 b). When the REVERSE transcription reaction and Cas13a were mixed in one single tube and cDNA yields were followed using qPCR, the amount of cDNA in the REVERSE group was comparable to the crRNA-free control, approximately 10-fold higher than the level of cDNA produced in the SHINE group (fig. 1 c). Taken together, these data indicate that Cas13a does not interfere with the REVERSE transcription reaction and amplification steps of the substrate in REVERSE.
Comparison of the two methods REVERSE and SHINE to detect SARS-CoV-2
Experiments have shown that REVERSE reaches the maximum fluorescence value faster than she, about 10 minutes faster, and that the signal value is 2.5 times that of she (fig. 1 d). To directly compare the limit of detection between REVERSE and SHINE, both one-step methods were performed under the same conditions, including the same input volume, sample concentration, reaction volume, and reaction time. REVERSE was able to detect 1 cp/. Mu.L of in vitro transcribed RNA samples 100-1000 fold more sensitive than SHINE (100-1000 cp/. Mu.L) (FIG. 1 e). A similar trend was also observed when using the extracted pseudoviral SARS-CoV-2N gene RNA as substrate (FIG. 1 f). When REVERSE is used to detect SARS-CoV-2 clinical samples, it can accurately detect all 17 SARS-CoV-2RNA samples with Ct values ranging from 29.1 to 35.9 (FIG. 1 g).
Feasibility assessment of reverse at lower temperatures
Antigen detection is performed at room temperature and is widely used. CRISPR detection methods typically require high temperature reactions, ranging in temperature from 37-60 ℃. Although CRISPR-based one-step detection is fast and sensitive, the need for heating equipment increases costs, limiting its widespread use. In order to evaluate the feasibility of REVERSE at low temperatures, the present invention tested REVERSE reactivity at 23 ℃ to 37 ℃. Experiments have shown that REVERSE is able to detect SARS-CoV-2RNA within 45 minutes at 23℃C (FIG. 2 a).
REVERSE optimization
In order to accelerate the reaction rate of REVERSE at room temperature, the present invention optimizes REVERSE. To achieve this objective, the present invention adjusts the concentration of the primer set (1-fold, 2-fold, 3-fold and 4-fold increase in reverse transcription primer TTTTGGTGTATTCA, RPA forward primer CCTCGAGGACAAGGCGTTCCAATTAA and reverse primer CCGGTAATACGACTCACTATAGGGCCCATATGATGCCGTCTTTG) and the fluorescent reporter gene (1-fold, 2-fold, 3-fold and 4-fold increase in reporter gene 5 '-FAM-uuuuu-BHQ 1-3') (fig. 2 b-c). Increasing the combination of 2-fold primer set dose and 3-fold fluorescent reporter dose significantly accelerated the response rate of REVERSE, enabling a significant fluorescent signal to be generated within 30 minutes at 20 ℃ (fig. 2 d). The optimized REVERSE of the present invention, referred to as REVERSE-2, was effective in detecting low concentrations of SARS-CoV-2RNA (0.5-5 cp/. Mu.L) within 30 minutes at room temperature at 20 ℃ (FIGS. 2 e-f).
Suitability assessment of REVERSE-2 for detection of clinical SARS-CoV-2 samples at 20 ℃
The invention tested 87 samples of SARS-CoV-2 positive nasopharyngeal swabs, ct values ranging from 22.2 to 37.6, and 20 negative samples. REVERSE-2 exhibited a sensitivity of 98.9% and a specificity of 100%, and a clinical sample of SARS-CoV-2 with a Ct value of 37.6 (equivalent to 0.5 cp/. Mu.L) could be detected within 30 minutes at room temperature (FIG. 2 g-k). Notably, the positive fluorescent signal can be read by a simple blue light device, and this result can be captured by a smartphone and camera, a feature that allows REVERSE-2 to be used as a simple self-test kit for self-test and bedside diagnostics (FIGS. 2 i-j).
6. The detection capabilities of Cas13a, cas12a and Cas12 b-mediated CRISPR one-step methods at room temperature were compared
The invention tests the effect of Cas13a, cas12a and Cas12b mediated CRISPR one-step detection at 20 ℃ or 25 ℃. The results indicate that only REVERSE-2 can generate a significant signal within 30 minutes at 20 ℃, while Cas12a can only generate a very weak signal at 25 ℃, and Cas12b cannot generate any signal at both temperatures (fig. 3 a). This observation suggests that Cas13a is the best CRISPR-associated protein to establish a room temperature one-step approach.
7. Ability to detect SARS-CoV-2 by comparing REVERSE-2 with commercial antigen at room temperature
The invention directly compares the speed, limit of detection and specificity of REVERSE-2 and commercial antigen detection SARS-CoV-2 clinical samples at room temperature. The reaction time for antigen detection was 15 minutes, with a limit of detection corresponding to a Ct value of 27.5 in RT-qPCR. In contrast, REVERSE-2 has a limit of detection corresponding to a Ct value of 37.3 in RT-qPCR, showing a sensitivity of about 500-1000 times that of antigen detection (FIGS. 4 a-b). Neither of these two assays showed cross-reactivity with herpes simplex virus type 1 (HSV-1) and Human Cytomegalovirus (HCMV), indicating high specificity for both assays (FIGS. 4 c-d). The result shows that the detection limit of REVERSE-2 is equivalent to that of RT-qPCR, the accuracy of the detection result is ensured, and the application potential is higher than that of antigen detection.
Example 2
The main materials involved in this example are as follows: reverse transcriptase (RNA-dependent DNA polymerase) was purchased from companies such as erbitux, sammer and lucigen, the recombinase polymerase amplification kit was purchased from the future company of the eastern city, cas13 protein and T7RNA polymerase were purified by the escherichia coli expression system, RNaseH was purchased from New EnglandBiolabs, RNA enzyme inhibitor was purchased from the company of the nonvozan and Promega Corporation, single-stranded RNA reporter gene was synthesized from the company of the large even treasures, rtp was purchased from the company Sigma, and primers were synthesized from the company of the marchan biology and the company of the marchan department of the biology.
The preparation of REVERSE-2 reaction liquid specifically comprises the following steps:
(1) Preparing pathogen lysis solution: to the lysis solution was added tris-2-chloroethyl phosphate at a final concentration of 250. Mu.M, 0.02. Mu.g. Mu.L -1 Chelex-100 and 1 U.mu.L -1 RNasin Plus。
(2) Mixing the lysate with pathogen sample at 1:1, and treating at 95deg.C for 10 min or at 65deg.C for 20-30 min.
(3) Preparing 30 mu LREVERSE-2 reaction liquid, wherein the REVERSE-2 reaction liquid contains a final concentration of 50nM LwaCas13a,22.5nM crRNA,1U/mu L RNase inhibitor, 1.5-2.5U/mu L REVERSE transcriptase, 0.05U/mu L RNase H,2mM of each rNTP,90 ng/mu L T RNA polymerase, 140nM FAM-UUUU-BHQ 1, 62.5nM RPA REVERSE primer, 250nM RPA forward primer, 185nM REVERSE transcription primer, 1 human recombinant enzyme polymerase reagent, 2 mu L of a cleaved sample or extracted RNA sample, and 40% of one-tube RPA lyophilized powder.
(4) The reaction mixture of REVERSE-2 was allowed to stand at 37℃for 15-30 minutes.
(5) Detecting by an enzyme-labeled instrument or an ultraviolet lamp or a blue light lamp.
The method for detecting target RNA by using REVERSE at room temperature is specifically as follows:
(1) Preparing pathogen lysis solution: to the cleavage solution was added tris (2-chloroethyl) phosphate at a final concentration of 250. Mu.M, 0.02. Mu.g/. Mu.L -1 Chelex-100, and 1 U.mu.L -1 RNasin Plus。
(2) Mixing the lysate with pathogen sample at 1:1, and treating at 95deg.C for 10 min or at 65deg.C for 20-30 min.
(3) A30. Mu.L one-step system was prepared containing 50nM LwaCas13a,22.5nM crRNA,1U/. Mu.L RNase inhibitor, 1.5-2.5U/. Mu.L reverse transcriptase, 0.05U/. Mu.L RNase H,2mM ofeach rNTP,90ng/. Mu. L T7RNA polymerase, 420nM FAM-UUU-BHQ 1, 125nM RPA reverse primer, 500nM RPA forward primer, 370nM reverse transcription primer, 1 human recombinase polymerase reagent, 2. Mu.L RNA substrate in 40% one-tube RPA lyophilized powder.
(4) The reaction mixture of REVERSE-2 was left at room temperature for 15-30 minutes.
(5) Target RNA as low as 0.5 cp/. Mu.L can be detected within 30 minutes at 20 ℃ by detection with an enzyme-labeled instrument or an ultraviolet or blue light.
The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A composition for detecting RNA at room temperature comprising:
reverse transcription primer and reverse transcriptase of target RNA, which are used for synthesizing reverse complementary sequence of target RNA, the length of the reverse transcription primer is 10-20 nucleotides;
the method comprises the steps of (1) an RPA reverse primer, an RPA forward primer, a recombinase, a DNA polymerase and a T7RNA polymerase, wherein the RPA reverse primer, the recombinase, the DNA polymerase and the T7RNA polymerase are used for amplifying a double-stranded DNA amplicon of target RNA, and synthesizing long-chain RNA containing a reverse complementary sequence of the target RNA by taking the double-stranded DNA amplicon as a substrate; the double-stranded DNA amplicon is formed by base complementary pairing of target RNA and a reverse complementary sequence thereof, and the 5' end of the RPA reverse primer comprises a T7RNA polymerase promoter sequence;
a Cas13a protein and crrnas thereof, the crrnas being for targeted recognition of a reverse complement of a target RNA, the Cas13a protein being for cleavage of the reverse complement of the target RNA;
TaqMan fluorescent probes, which can be nonspecifically cleaved by Cas13a protein when the reverse complement of the target RNA is cleaved, release the fluorescent group.
2. The composition for detecting RNA at room temperature of claim 1, wherein:
the reverse transcription primer is 12-16 nucleotides in length.
3. The composition for detecting RNA at room temperature of claim 1, wherein:
the Cas13a protein is at least one of lwcas 13a, lbasa 13a, lshCas13a, pprCas13a, ereCas13a, lneCa3a, camCas13a, rcaCas13a, hheacas 13a, lbuCas13a, lseCas13a, lbmCas13a, lbnncas 13a, rccas 13a, rcraccas 13a, rccdcas 13a, cgCas13a, cg2Cas13a, lwcas 13a, lbfCas13a, lba4Cas13a, lba9Cas13a, lneCas13a, hheacas 13a, rcaCas13 a.
4. The composition for detecting RNA at room temperature of claim 1, wherein:
the concentration of the reverse transcription primer of the target RNA is 100-1000nM; the concentration of the RPA reverse primer is 50-500nM; the concentration of the RPA forward primer is 150-1000nM.
5. The composition for detecting RNA at room temperature of claim 1, wherein:
the concentration of TaqMan fluorescent probe is 100-1000nM.
6. The composition for detecting RNA at room temperature of claim 1, wherein:
the fluorescence quenching group of the TaqMan fluorescent probe is BHQ, BHQ1, BHQ2 or TAMRA, and the fluorescence reporting group is FAM, CY3, CY5, HEX or TET.
7. The composition for detecting RNA at room temperature of claim 1, wherein:
the target RNA is SARS-CoV-2RNA, and the sequence is ACUGAGAUCUUUCAUUUUACCGUCACCA;
the crRNA is GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUGGUGACGGU AAAAUGAAAGAUCUCAGU;
the sequence of the reverse transcription primer of the target RNA is as follows: TTTTGGTGTATTCA;
the sequence of the RPA reverse primer is:
CCGGTAATACGACTCACTATAGGGCCCATATGATGCCGTCTTTG；
the sequence of the RPA forward primer is: CCTCGAGGACAAGGCGTTCCAATTAA;
the RNA sequence of the TaqMan fluorescent probe is at least one of UUUUU, UUUUUU, UUUUUUU, UUUUUUUU.
8. The composition for detecting RNA at room temperature of claim 1, wherein:
the room temperature nucleic acid detection kit also comprises an RNase inhibitor, an RNase H, ribonucleoside triphosphates, deoxyribonucleoside triphosphates, a buffer solution and a reaction activator.
9. A room temperature nucleic acid detection kit, characterized by comprising the composition for detecting RNA at room temperature according to any one of claims 1 to 8.
10. The room temperature nucleic acid detection kit of claim 9, wherein:
the room temperature nucleic acid detection kit comprises: 20-200nM LwaCas13a or LbuCas13a,10-100nM crRNA, 1.5-2.5U/. Mu.L reverse transcriptase, 50-500 ng/. Mu.L T7RNA polymerase, 100-1000nM TaqMan fluorescent probe, 50-500nM RPA reverse primer, 150-1000nM RPA forward primer, 100-1000nM reverse transcription primer.
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| CN112239795A (en) * | 2020-09-16 | 2021-01-19 | 复旦大学 | RT-RPA and CRISPR-based visual new coronavirus RNA nucleic acid detection kit |
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