CN115820818B - One-step method nucleic acid detection method and application thereof - Google Patents
One-step method nucleic acid detection method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a one-step method for detecting nucleic acid and application thereof. In the invention, in the same space, a reaction system for amplifying RAA at constant temperature by DNA and a CRISPR/Cas12b fluorescent signal detection system for gene editing are added simultaneously, so that the nucleic acid detection can be realized without extra amplification product transfer steps, the practical operation is simple and quick, and aerosol pollution is avoided. The nucleic acid detection method provided by the invention can reduce the whole process from sample to nucleic acid result interpretation to within 30min, and the system does not need to extract nucleic acid, has the sensitivity of 0.2 copies/mu L, has extremely high sensitivity, can be used for detecting the nucleic acid of the bordetella pertussis, and is beneficial to reducing the infection rate and the disease transmission rate.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a one-step method for detecting nucleic acid and application thereof.
Background
Viral or bacterial infection of the respiratory tract is one of the most common diseases in humans worldwide, and identification of the causative agent of respiratory viral infection is of great importance in the selection of appropriate treatments and prevention of epidemics. Therefore, the development of a reagent for rapidly and accurately diagnosing pathogens has profound effects on disease monitoring, drug guidance and treatment evaluation.
Pertussis is an acute respiratory tract infection caused by bordetella pertussis, and early diagnosis is important for treating pertussis. Currently, common pertussis diagnostic methods in the laboratory include culture, direct fluorescent antibody detection, PCR, pairing and single serological techniques (a.m. Wendelboe and a.van Rie, expert review of molecular diagnostics, 2006, 6, 857-864.). Isolation of bordetella pertussis by culture is a traditional "gold standard" for diagnosis of pertussis. Its specificity was about 100%. However, the sensitivity of the culture is low, varying from 12% to 60%, and the culture takes a long time, on average 5 days to 10 days. Traditional Polymerase Chain Reaction (PCR) or reverse transcription polymerase chain reaction (RT-PCR) methods are gold standard for nucleic acid detection and have high sensitivity. Since the 90 s of the 20 th century, PCR has been used as a diagnostic tool for clinical bordetella pertussis and is widely used in research settings. However, PCR technology requires a skilled operator and a well-established equipment infrastructure, and sample detection times are long, which is not suitable for simple and rapid point of care testing (POCT).
In recent years, nucleic acid detection methods based on regularly clustered short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) have become a hotspot for point of care detection (POCT). RNA-guided CRISPR/Cas system proteins not only cleave cis (cis-clean) target nucleic acids, but also can cleave non-specifically trans (trans-clean) flanking single-stranded non-target nucleic acids when they recognize the target nucleic acid. By taking advantage of this property, CRISPR/Cas nuclease proteins are combined with isothermal amplification techniques, such as Recombinase Polymerase Amplification (RPA) and loop-mediated isothermal amplification (LAMP), researchers have developed a series of sensitive, rapid and visualized nucleic acid detection methods that do not require special precision instrumentation. For example, specific high sensitivity shorlock based on Cas13a or Cas13 b; the Cas12 a-based DNA endonuclease targets the detect of CRISPR and Cas12 b-mediated DNA detection CDetection based on Cas12 b. However, these methods require a two-step process of amplifying the target nucleic acid sequence followed by CRISPR detection. Such multiple processing steps increase operating time, cross-contamination and risk of aerosol generation.
Disclosure of Invention
Aiming at the technical problems, the invention provides a one-step method nucleic acid detection method, which can simultaneously add two reaction systems in the same space without additional amplification product transferring steps, has simple and quick actual operation, avoids aerosol pollution and avoids cross contamination.
In order to achieve the technical effects, the invention adopts the following technical scheme:
a one-step nucleic acid detection method comprising the steps of:
s1, mixing and vibrating a target detection sample and a release agent to obtain a mixture I;
s2, simultaneously adding a DNA isothermal amplification system RAA containing a specific primer designed for a target sequence into a PCR reaction tube, and a gene editing CRISPR/Cas12b system containing a specific sgRNA designed for the target sequence and a fluorescent reporter probe;
s3, adding a detection sample and a mixture I detection sample of a release agent into the reaction system, carrying out vortex oscillation and short-term centrifugation (instantaneous centrifugation by a palm centrifuge) to obtain a mixture II;
s4, placing the mixture II obtained in the step S3 into a PCR instrument for reaction, synchronously reading fluorescence, and judging the result according to the fluorescence signal to obtain the fluorescent material.
Preferably, the PCR reaction tube in step S2 may also be a isothermal heater with a read fluorescent signal, a isothermal nucleic acid amplification apparatus, or an end point fluorescent reading under an LED blue or ultraviolet lamp after the isothermal heater is placed.
The equipment matched with the reaction system is not limited to a PCR instrument, but also comprises a constant temperature heater or a constant temperature nucleic acid amplification instrument for reading fluorescent signals, or the equipment is placed into the constant temperature heater at 37 ℃ for 30min and then subjected to end point fluorescence interpretation under an LED blue light or ultraviolet lamp.
Preferably, the DNA isothermal amplification system RAA of step S1 further comprises a molecular crowding reagent, a nucleic acid amplification starter, a reaction dry powder enzyme preparation, and an amplification primer.
Preferably, the molecular crowding agent is polyethylene glycol of 20000 (including but not limited to); the nucleic acid amplification starter is 280mM magnesium acetate (including but not limited to).
Preferably, the gene editing CRISPR/Cas12b system of step S2 further comprises a Cas12b enzyme, an sgRNA, and a fluorescent probe.
Preferably, the fluorescent probe is provided with a fluorescent group and a quenching group.
Preferably the fluorophore is FAM (including but not limited to); the quenching group is BHQ1 (including but not limited to); the fluorescent probe is FAM-CCCCC-BHQ 1.
Preferably, the reaction process in the step S4 is carried out at the temperature of 30-42 ℃ for 25-35 min.
The invention also provides application of the detection method in preparation of a pertussis detection kit.
The invention also provides a bordetella pertussis detection kit, which comprises a DNA isothermal amplification RAA system, a gene editing CRISPR/Cas12b fluorescent signal detection system and a use instruction.
In the invention, we provide a one-step method nucleic acid detection method Rcod, wherein DNA isothermal amplification RAA and gene editing CRISPR/Cas12b fluorescent signal detection are carried out simultaneously in one step, no extra amplification product transfer step is needed, and the practical operation is simple and quick, and aerosol pollution is avoided. Furthermore, the one-step nucleic acid detection method Rcod provided by the invention has universality and can be used for detecting cells, viruses, bacteria and the like containing target nucleic acid.
The inventors first explored the trans-cleavage activity (trans-cleavage activity) and cis-cleavage activity (cis-cleavage activity) of CRISPR/Cas12b, respectively, alone over an extremely broad temperature range of 25 ℃ -72 ℃. It was confirmed that CRISPR/Cas12b exhibited extremely strong trans-cleavage activity in the temperature range of 30-68 ℃, whereas CRISPR/Cas12b had extremely strong cis-cleavage activity in the temperature range of 45-60 ℃. The optimal reaction temperature range of 30-42 ℃ for further combining DNA isothermal amplification RAA is finally determined, the optimal reaction temperature range of 30-42 ℃ for one-step nucleic acid detection is determined, and the acidophilic alicyclic bacillus Cas12b (AacCas 12 b) still has higher trans-cleavage activity in the temperature range, so that a specific cleavage probe can be used, and the whole reaction system has extremely strong specificity; the cis-form cutting activity is extremely weak, so that the DNA isothermal amplification product is not excessively cut, and the sensitivity of the one-step nucleic acid detection system is ensured. The nucleic acid detection method provided by the invention can reduce the whole process from sample to nucleic acid result interpretation to within 30min, and the system does not need to extract nucleic acid, and the sensitivity reaches 0.2 copies/. Mu.L. We examined 221 clinical samples of Bordetella pertussis, and compared with the PCR detection results, the sensitivity and specificity of the invention were 97.96% and 99.19%, respectively.
The one-step method nucleic acid detection method Rcod provided by the invention does not need an extra amplification product transferring step, is simple and quick in actual operation, and avoids aerosol pollution.
The primer pair used in the invention IS obtained by screening a large number of sgRNA and probes, has strong detection specificity and high sensitivity on IS1663 sequences of the bordetella pertussis, and the detection lower limit IS 0.2 DNA copies per microliter. The invention adopts RAA-CRISPR/Cas12b technology to establish a one-step method for rapidly detecting the pertussis bacillus, has high sensitivity and high specificity of molecular biological detection, is simple and convenient to operate, does not depend on complex instruments, and completely meets the requirements of bedside detection of the pertussis bacillus.
The technical scheme of the invention can obviously improve the detection speed. Compared with the conventional PCR, the RAA-CRISPR/Cas12b reaction is carried out synchronously at 37 ℃ without three steps of denaturation, annealing and extension, and the PCR can be completed within 30 minutes. The detection method does not need complicated instruments and equipment, is not easy to pollute aerosol, and is suitable for on-site detection. The detection method can amplify visual end point fluorescence detection under the normal temperature isothermal condition, does not need to rely on complex instruments and equipment such as a PCR instrument, a fluorescence quantitative PCR instrument, an electrophoresis tank and the like, does not need complex sample treatment, can release sample nucleic acid by using a releasing agent, can automatically detect by any person according to a specification, and can truly realize portable on-site rapid nucleic acid detection.
The invention establishes the kit for rapidly detecting the pertussis bacillus by adopting the one-step RAA-CRISPR/Cas12b technology for the first time, can be used for clinical field detection by specificity, sensitivity and actual sample analysis, and provides a sensitive and reliable novel method for the instant detection of the pertussis bacillus.
Compared with the prior art, the nucleic acid detection method provided by the invention is carried out by two reaction systems simultaneously in one step. The kit comprises a reaction system I for amplifying RAA at a constant temperature by DNA, and a reaction system II for detecting CRISPR/Cas12b fluorescent signals by gene editing. The nucleic acid detection method provided by the invention can simultaneously add two reaction systems in the same space, does not need an additional step of transferring amplification products, is simple and quick in actual operation, and avoids aerosol pollution. The nucleic acid detection method provided by the invention can reduce the whole process from sample to nucleic acid result interpretation to within 30min, and the system does not need to extract nucleic acid, has the sensitivity of 0.2 copies/mu L, and has extremely high sensitivity. The nucleic acid detection method provided by the invention has the characteristics of simplicity in operation, no aerosol pollution, no toxic substances, short reaction time, high reaction sensitivity and the like, can greatly meet the POCT (point of care testing) requirements of the bordetella pertussis at the bedside, is beneficial to early diagnosis and early treatment, and reduces the infection rate and the disease transmission rate.
Drawings
FIG. 1 is a schematic diagram showing the structure of one embodiment of the one-step nucleic acid detection method of the present invention;
FIG. 2 shows the fluorescence signal results of the one-step nucleic acid detection method Rcod of the present invention under different temperature conditions;
FIG. 3 is a diagram showing the result of detecting the lowest limit of detection of bordetella pertussis by using the one-step nucleic acid detection method Rcod of the present invention;
FIG. 4 is a graph showing the results of detection of pertussis clinical samples using the one-step nucleic acid detection method Rcod of the present invention.
Detailed Description
The technical scheme of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only for illustrating the technical scheme of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical scheme of the present invention may be modified or substituted without departing from the spirit and scope of the technical scheme of the present invention, and all such modifications are intended to be included in the scope of the claims of the present invention.
Example 1 one-step nucleic acid detection method
As shown in FIG. 1, the one-step nucleic acid detection method Rcod of the present invention includes its use for DNA isothermal amplification RAA and for gene editing CRISPR/Cas12b fluorescent signal detection. In the same reaction system, selecting target sequences according to different target nucleic acid molecules to be detected in each target detection object sample, designing corresponding specific primers aiming at the target sequences of the target nucleic acid molecules to be detected in each target detection object sample, and adding a release agent to the target detection sample as a target nucleic acid template; meanwhile, selecting target sequences according to different target nucleic acid molecules to be detected in each target detection object sample, and designing corresponding sgRNA aiming at the target sequences, wherein a single-chain base sequence with a fluorescent group and a quenching group is used as a fluorescent report probe.
1.1. Design of primers and sgRNA
The IS1663 gene of the pertussis bacillus (the sequence ID IS CP 046995.1) (the gene sequence can be used as a conserved sequence) IS adopted as a target sequence, and specific primers for amplifying the target sequence are as follows:
the upstream primer FP: CCTACGGGTCTGTATCACGAGCAAGCGGC;
downstream primer RP: GCGTCTGTCCATAGCGAGCCAGCACGTAGC;
specific sgrnas (aacas 12 b) are:
GGUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCCGUUGAGCUUCUCAAAUCUGAGAAGUGGCACCCAAGGCCUUGGCGCUGCGC；
nonspecific fluorescent probe P: FAM-CCCCC-BHQ 1.
1.2. Extraction of pathogen DNA
The nasopharyngeal swab is placed in 1mL of 0.9% physiological saline for eluting, and then the eluent is taken and mixed with an equal volume of release agent.
The pathogen DNA is extracted without a special nucleic acid extraction kit, 20 mu L of target detection sample and release agent (the main components are NaOH, tris-acetic acid pH8.0, dextran, mannitol, sucrose and the like, the addition amount is 20 mu L) are subjected to instantaneous centrifugation on a palm centrifuge, and mixed and vibrated;
the release agent is added according to the following formulation: 2M NaOH5mL; 5mL of 1M Tris-acetate at pH 8.0; 2.5mL of dextran with the mass concentration of 20%; mannitol with a mass concentration of 12.5% of 5mL; 2.5mL of sucrose with the mass concentration of 20%; adding water to fill up to 50mL, mixing uniformly and preserving at normal temperature for standby.
3. Configuration of one-step nucleic acid detection system
The PCR reaction tube was simultaneously charged with the upstream primer FP (10. Mu.M, 0.8. Mu.L in addition), the downstream primer RP (10. Mu.M, 0.8. Mu.L in addition), specific sgRNA (AacCas 12b, 0.3. Mu.g/. Mu.L, 1. Mu.L), non-specific fluorescent probe P (25 mM, 1. Mu.L) in step 2, 6.25. Mu.L (which may be but is not limited to 20,000 polyethylene glycol (PEG)), 1.25. Mu.L (which may be but is not limited to 280mM magnesium acetate) of nucleic acid amplification starter, a reaction dry powder enzyme preparation (available from Hangzhou Biotech Co., ltd., RAA lyophilized reagent enzyme, one system of which was added half-tube) and Cas12b enzyme (2 mg/mL, 1. Mu.L) in a concentration of 6.25% molecular crowding reagent, 1.25. Mu.L, and water to 20. Mu.L.
Then 5 mu L of the detection sample mixed with the release agent is taken and added into the reaction system, vortex vibration and short centrifugation are carried out;
the mixed one-step reaction system is put into a PCR instrument, the temperature is set at 37 ℃, and the reaction time is 30min.
4. Experimental results
The real-time fluorescence reading can be carried out within 30min of the reaction, or the end point fluorescence interpretation can be carried out under an LED blue light or ultraviolet lamp after 30min, and the result interpretation can be carried out according to the fluorescence signal.
Example 2 optimal reaction temperature selection
Since aacas 12b in example 1 shows weak cis-cleavage activity at a lower temperature, this example tests the optimal reaction temperature of the one-step nucleic acid detection method rcd established in example 1 at a temperature of 30 ℃ to 45 ℃. pUC57-IS1663 plasmid containing the pertussis IS1663 sequence was diluted with TE buffer to a concentration of 200 copies/. Mu.L for use as a target sequence.
As shown in FIG. 2, the whole system was incubated at 37℃for 10min, and then the temperature was raised to 48℃for 20min (37-48 ℃); or respectively reacting at 30deg.C, 32deg.C, 35deg.C, 37deg.C, 39deg.C, 42deg.C and 45deg.C for 30min.
As a result, FIG. 2a shows the real-time fluorescence signal monitored within 30min of the reaction, and FIG. 2b shows the endpoint fluorescence detection after 30min of the reaction. As can be seen from fig. 2, the one-step nucleic acid detection method rcd shows significantly enhanced fluorescence signal at a temperature of 32 to 39 ℃. In addition, the fluorescence signal of the reaction was almost consistent when compared with the control group (amplification was performed by a reaction at 37℃for 10min and CRISPR fluorescence detection was performed by a reaction at 48℃for 20 min) after a constant temperature reaction at 37 ℃. After incubation for 30min, the endpoint fluorescence signal showed consistent results by either LED blue light irradiation or uv light irradiation. Therefore, we selected 37℃as the optimal reaction temperature for a one-step nucleic acid detection system.
Example 3 lowest limit of detection LOD test
This example performs a minimum limit of detection LOD test on one-step nucleic acid detection method Rcod for the amplification and detection of bordetella pertussis established in example 1. The method comprises the following steps:
a series of 10-fold dilutions of the bordetella pertussis IS-1663 plasmid (i.e., pUC57-IS1663 in example 2) were used as templates, and 4 replicates were performed for each gradient. Also diluted with TE buffer, the concentration of target sequence templates was 2X 10, respectively 3 、2×10 2 、2×10 1 、2×10 0 、2×10 -1 And negative control NTC. The real-time fluorescence signal was monitored during 30min of reaction (fig. 3 a), after 30min of reaction, the endpoint fluorescence detection was performed (fig. 3 b), and after 30min of reaction, the endpoint fluorescence photographing was performed by LED blue light irradiation or ultraviolet light irradiation.
As can be seen from FIG. 3, the real-time fluorescence and the LED blue light/ultraviolet end point fluorescence result are consistent, showing that the one-step nucleic acid detection system can stably detect the input plasmid DNA as low as 0.2 copies/. Mu.L at 37 ℃, and has extremely high sensitivity.
Example 4 clinical test
This example truly tests a clinical sample of a one-step nucleic acid detection method rcd for the amplification and detection of bordetella pertussis established in example 1. The method comprises the following steps:
in order to verify the clinical applicability of the Rcod platform, 98 pertussis culture positive samples and 123 pertussis culture negative samples of Beijing children hospitals affiliated to the university of capital medical science were tested by a one-step nucleic acid detection method Rcod and a real-time fluorescence quantitative PCR method. The infection status of the patient is independently confirmed by Beijing children hospitals affiliated to the university of capital medical science. The 221 samples were equally divided into two parts, one part was used for the one-step nucleic acid detection method rcd and the other part was used for real-time fluorescent quantitative PCR (these 2 methods all detect 221 samples). In addition, in order to facilitate rapid detection of clinical samples, the samples were treated using the nucleic acid releasing agent method (i.e., the samples to be tested of the present invention were mixed with a releasing agent), and sample extraction was completed within 5 seconds. Meanwhile, the pertussis nucleic acid detection kit (PCR fluorescent probe method) is used for detection; the kit can be purchased from Shenzhen Yi cubic biotechnology limited company, the production license number is Guangdong food and medicine monitoring instrument production license 20142667, the national instrument standard number is 20203400152, the latest approval date of the instruction book is 14 days of 2 months in 2020, and the real-time PCR test process is operated according to the latest approval instruction book.
As shown in fig. 4, wherein (a, b) total 221 pertussis clinical samples, (a) 98 positive samples, (b) 123 negative samples; (c) Fluorescence values of pertussis positive patient (+) and control (-). (d) Waterfall distribution Waterfall distribution (n=221) for all bordetella pertussis clinical samples. (e) And detecting the pertussis bacillus in the clinical sample, and directly visualizing the detection result under the blue light and ultraviolet light of the LED.
FIGS. 4a-b show that we compare the raw fluorescence intensity of the one-step nucleic acid detection method Rcod with the cycle threshold (Ct) of real-time fluorescent quantitative PCR, which is proportional to the concentration of the target gene. Both detection methods showed good agreement. By our one-step nucleic acid detection method rcd, 97 pertussis positive culture samples were identified as bordetella pertussis positive with an initial fluorescence intensity greater than 2699a.u. (3 x average of initial fluorescence values for all samples) (fig. 4a, c-d). Furthermore, our one-step nucleic acid detection method rcd identified 124 pertussis culture negative samples as bordetella pertussis negative with endpoint fluorescence values less than 2000a.u. (fig. 4 b-d). In addition, the end point fluorescence detection under LED blue light irradiation or ultraviolet light irradiation showed consistent results (fig. 4 e).
In addition, we calculated the sensitivity and specificity of the one-step nucleic acid detection method Rcod for detection of clinical samples of B.pertussis. In comparison to real-time fluorescent quantitative PCR, our one-step nucleic acid detection method Rcod showed 97.96% sensitivity, 99.19% specificity and 98.64% accuracy, with a detection time of 30min, and a real-time fluorescent quantitative PCR of 2 hours (Table 1). The one-step nucleic acid detection method Rcod has greater application value in clinical diagnosis and detection. Can be used for clinical field detection, and provides a sensitive and reliable new method for the instant detection of the pertussis bacillus.
TABLE 1 comparison of the identity between the detection method of the invention and real-time PCR for 221 clinical samples
Finally, it should be noted that the above-mentioned embodiments are merely illustrative of the principles, performances and effects of the present invention, and are not meant to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (3)
1. A one-step nucleic acid detection method for non-diagnostic purposes, comprising the steps of:
s1, mixing and vibrating a target detection sample and a release agent to obtain a mixture I;
s2, simultaneously adding a DNA isothermal amplification system RAA containing amplification primers designed for a target sequence into a PCR reaction tube, and a gene editing CRISPR/Cas12b system containing specific sgRNA designed for the target sequence and a fluorescent reporter probe;
s3, adding the mixture I of the detection sample and the release agent into the reaction system, carrying out vortex oscillation and short-time centrifugation to obtain a mixture II;
s4, placing the mixture II obtained in the step S3 into a reaction instrument for reaction, reading fluorescence, and judging the result according to the fluorescence signal to obtain the fluorescent material; s2, the DNA isothermal amplification system RAA comprises a molecule crowding reagent, a nucleic acid amplification starter, a reaction dry powder enzyme preparation and an amplification primer; the molecular crowding reagent is polyethylene glycol with molecular weight of 20000; the gene editing CRISPR/Cas12b system in the step S2 contains Cas12b enzyme, sgRNA and fluorescent probe; the nucleic acid amplification starter is 280mM magnesium acetate; the amplification primer comprises an upstream primer FP and a downstream primer RP, wherein the specific sequence of the upstream primer FP is 5'-CCTACGGGTCTGTATCACGAGCAAGCGGC-3', and the specific sequence of the downstream primer RP is 5'-GCGTCTGTCCATAGCGAGCCAGCACGTAGC-3'; the specific sequence of the sgRNA is 5'-GGUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCA AAGCCCGUUGAGCUUCUCAAAUCUGAGAAGUGGCACCCAAGGCCUUGGCGCUGCGC-3'; the reaction process in the step S4 is that the reaction is carried out for 25-35 min at the temperature of 30-42 ℃; the release agent in the step S1 is added according to the following formulation materials: 2M NaOH5mL; 5mL of 1M Tris-acetate at pH 8.0; 2.5mL of dextran with the mass concentration of 20%; mannitol with a mass concentration of 12.5% of 5mL; 2.5mL of sucrose with the mass concentration of 20%; adding water to fill up to 50mL, uniformly mixing and preserving at normal temperature for later use; the fluorescent group is FAM; the quenching group is BHQ1; the fluorescent probe is FAM-CCCCC-BHQ 1.
2. The detection method according to claim 1, wherein the reaction instrument in the step S4 is one of a PCR reaction tube, a constant temperature heater for reading fluorescent signals and a constant temperature nucleic acid amplification instrument; the fluorescence reading method comprises the following steps: and directly and synchronously reading fluorescence according to the PCR reaction, or firstly placing the fluorescence in a constant temperature heater and then judging the fluorescence of the end point under the blue light or ultraviolet lamp of the LED.
3. The method of claim 1, wherein the fluorescent probe has a fluorescent group and a quenching group.
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| CN110904198A (en) * | 2019-12-11 | 2020-03-24 | 博迪泰（厦门）生物科技有限公司 | Nucleic acid one-step detection method based on constant-temperature amplification and gene editing |
| CN111635950A (en) * | 2020-05-28 | 2020-09-08 | 深圳市亿立方生物技术有限公司 | Kit and method for rapidly detecting bordetella pertussis |
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| CN115335536A (en) * | 2021-01-08 | 2022-11-11 | 武汉大学 | Compositions and methods for point-of-care nucleic acid detection |
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| CN109837328A (en) * | 2018-09-20 | 2019-06-04 | 中国科学院动物研究所 | Nucleic acid detection method |
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