CN111850099A - RPA kit, primers, probe and method for detecting varicella virus - Google Patents
RPA kit, primers, probe and method for detecting varicella virus Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to an RPA kit, primers, a probe and a method for detecting a varicella virus nucleic acid. The kit comprises an RPA reaction system, wherein the RPA reaction system comprises an RPA primer probe mixed solution, the RPA primer probe mixed solution comprises a primer pair and a probe, the nucleotide sequence of the primer pair is shown as SEQ ID No.1 and SEQ ID No.2, and the probe is shown as SEQ ID No.3+ FAM-dt + THF + BHQ1-dt + SEQ ID No.4+ C3. The kit can be used for detecting the varicella virus, and has the advantages of short reaction time, high detection speed, high sensitivity and high specificity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an RPA kit, primers, a probe and a method for detecting a varicella virus nucleic acid.
Background
The data disclose that Mouse pox (Mouse pox) caused by the Mouse pox virus (EV) is a highly contagious, devastating virulent infectious disease that is prevalent in groups of laboratory mice worldwide with a mortality rate of 96.6%. The disease is characterized by swelling of limbs, head and tail, necrosis and even toe falling off clinically, and is one of the most serious viral diseases harming mice. Marchal in England equals 1929, the patient was first found in the England laboratory rat group and was first reported in the next year; the rat pox epidemic situation of the Beijing central biological product research institute is firstly reported in 1951 in the Tangfei province of China; wuhuixing et al (1986) and Xubei et al (1989) performed serological epidemiological investigation on the groups of rats across the country, confirming that mousepox is sporadic in the groups of rats in China. At present, the disease widely exists in all countries of the world, particularly the England American method is the most serious, and the disease still occurs in China. Research shows that the disease is often epidemic in outbreak, has high fatality rate, often causes complete group elimination, seriously influences the production and breeding of experimental animals, and extremely seriously influences the smooth performance of animal experiments and the accuracy and repeatability of scientific research data. Research shows that under natural conditions, the varicella virus can infect mice of various ages, but the fatality rate of the suckling mice and the aged mice above 1 year old is higher, while the mice of 6-12 weeks old are lower, and the susceptibility of the mice of different strains is different; because the mouse pox virus can be recessive infected, the mouse does not show clinical symptoms, but the virus can be persistently existed in the body and reproduced, and the virus is continuously discharged from excrement to become a terrible infection source, so that the early diagnosis of the mouse pox is particularly important for detecting the recessive infected mouse.
In the prior art, the serology method and the PCR method are the most commonly used methods for detecting EV, however, the two methods either need expensive instruments and equipment, or skilled technicians, or have long detection period, so the two methods are not suitable for popularization and application in common laboratories or field diagnosis.
In 2006, a new isothermal gene amplification technology, Recombinase Polymerase Amplification (RPA), was invented, which can amplify a large amount of target gene fragments in vitro isothermally at 37-42 ℃ within 10-30 min; subsequently, the UK TwistDx company develops an RPA commercialized kit, and the research, the popularization and the application of an RPA technology are accelerated; compared with Polymerase Chain Reaction (PCR), PRA has the advantages of high amplification speed, constant reaction temperature, simple required instrument and the like, and is particularly suitable for rapid detection of DNA or RNA. At present, RPA has been developed and applied in the detection of viruses, chlamydia, mycoplasma, bacteria, parasites and other pathogens; the technology combines a primer with a recombinase to form a protein-DNA mixture, and starts to search for a homologous sequence on a template DNA; when the homologous sequence is positioned, a strand displacement reaction is initiated; the primers are combined on the corresponding templates, and the polymerase starts to start DNA synthesis from the 3' end of the primers, so that the geometric series amplification of the two primers to the target gene is realized at normal temperature; meanwhile, in an RPA reaction system, the method has stronger tolerance to primer base mutation, and the RPA reaction can still be smoothly carried out after the individual base mutation of the primer.
Practice shows that recombinase polymerase amplification is a nucleic acid detection technology capable of replacing PCR, and single molecule nucleic acid detection can be carried out at normal temperature within 20 minutes; the technology has low requirements on hardware equipment, and is particularly suitable for being used in the field and quarantine sites.
Based on the foundation and the current situation of the prior art, in order to enhance the monitoring of the entry dental experiment animal and the wild animal for the mousepox epidemic situation of the port and the epidemiological investigation, the application provides the RPA kit, the primers, the probe and the method for detecting the mousepox virus on the basis of establishing the real-time fluorescence RPA EV detection method and verifying the specificity, sensitivity and stability of the method.
Disclosure of Invention
The invention aims to overcome the defects of low sensitivity and sensitivity, long reaction time and the like of the conventional detection of the varicella virus, and provides a Recombinase Polymerase Amplification (RPA) kit, a primer, a probe and a method for detecting EV nucleic acid based on a method for establishing real-time fluorescence RPA and a basis for verifying the specificity, sensitivity and stability of the method. The rapid detection method for detecting EV nucleic acid established by RPA has high specificity and sensitivity, and provides a rapid, simple, convenient and accurate detection method for diagnosing EV infection.
The invention mainly solves the technical problems in the prior art through the following technical scheme.
The invention provides a kit for detecting mouse pox virus (EV) nucleic acid, which comprises an RPA reaction system, wherein the RPA reaction system comprises an RPA primer probe mixed solution, the RPA primer probe mixed solution comprises a primer pair and a probe, the nucleotide sequence of the primer pair is shown as SEQ ID No.1 and SEQ ID No.2, and the probe is shown as SEQ ID No.3+ FAM-dt + THF + BHQ1-dt + SEQ ID No.4+ C3.
In order to improve the amplification efficiency, the concentration of the primers of the RPA reaction system is preferably 5-20 μ M, and more preferably 10 μ M; the concentration of the probe is preferably 5 to 10 μ M, and more preferably 10 μ M.
Preferably, the RPA reaction system further comprises a core reaction solution, a reaction buffer solution, an Exo reaction solution, a probe enzyme mixture, magnesium acetate and dNTP; wherein the concentration of the magnesium acetate is preferably 260-300 mu M, and more preferably 280 mu M. The concentration of the dNTP is preferably 20-220 μ M, and more preferably 200 μ M.
In a preferred embodiment of the present invention, the primer probe mixture comprises the primer pair and the probe, wherein the amount of each primer in the primer pair is 2.1. mu.L and the concentration is 10. mu.M, and the amount of the probe is 0.6. mu.L and the concentration is 10. mu.M. The amount of the core reaction solution is 2.5. mu.L, the amount of the reaction buffer solution is 25. mu.L, the amount of the probe enzyme mixture is 5. mu.L, and the amount of the Exo reaction solution is 1. mu.L; the amount of the magnesium acetate is 2.5 mu L, and the concentration is 280 mu M; the amount of dNTP was 7.2. mu.L, and the concentration was 11 mM. Wherein:
The core reaction solution is preferably 20 times the amount of the core reaction solution (i.e., 20 Xthe amount of the core reaction solution; the same applies hereinafter).
The reaction buffer is preferably 2 times the reaction buffer.
The probe enzyme mixture is preferably 10 times of the probe enzyme mixture;
the Exo reaction solution is preferably 50 times of Exo reaction solution
Preferably, in order to determine the detection result more intuitively and accurately, the kit further comprises a negative control, and the negative control is preferably ddH2O。
The invention also provides a primer pair for detecting the nucleic acid of the varicella virus (EV), wherein the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO.1 and SEQ ID NO.2 in the sequence table; preferably, the detection is RPA detection.
The invention also provides a probe for detecting the nucleic acid of the mousepox virus (EV), wherein the probe is shown as SEQ ID NO.3+ FAM-dt + THF + BHQ1-dt + SEQ ID NO.4+ C3; preferably, the detection is RPA detection.
The invention also provides a combination for detecting the nucleic acid of the varicella virus (EV), which comprises a primer pair and a probe, wherein the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO.1 and SEQ ID NO.2 in the sequence table; the probe is shown in SEQ ID NO.3+ FAM-dt + THF + BHQ1-dt + SEQ ID NO.4+ C3; preferably, the detection is RPA detection.
The invention also provides an application of the primer pair and/or the probe or the combination in preparing a kit for detecting the varicella virus (EV).
The invention also provides a method for detecting the varicella virus (EV) for non-diagnostic purposes, which comprises the following steps:
(1) extracting total DNA in a sample to be detected by using a DNA extraction reagent;
(2) taking the total DNA extracted in the step (1) as a template, and carrying out an RPA reaction by using an RPA reaction system in the kit, wherein the time of an amplification reaction in the RPA reaction is preferably 25min, and the temperature is preferably 39 ℃;
(3) and analyzing the detection result.
The method is used for respectively detecting other viruses of mice, such as Mouse Hepatitis Virus (MHV), Mouse Parvovirus (MPV), mouse Sendai Virus (SV), mouse reovirus type III (Reo-3), mouse parvovirus (MVM) and lymphocyte choriomeningitis virus (LCMV) as controls, and is compared with a PCR method to detect the specificity of the established method, and the result proves that the RPA detection method of the mousepox virus (EV) established by the invention has high specificity and sensitivity, and provides a quick, simple, convenient and accurate detection means for diagnosing SIV infection in a common laboratory of a basic unit.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The invention has the beneficial effects that:
the invention provides a novel RPA method for detecting mouse pox virus (EV) nucleic acid, compared with the common PCR method, the RPA has the following advantages: firstly, RPA belongs to an isothermal amplification technology, has low requirements on instruments and equipment, and can complete reaction only by a constant-temperature water bath kettle; secondly, the RPA detection speed is high, the reaction time is within 40min, and the reaction time is obviously shorter than that of the conventional PCR (usually 1-2 h); thirdly, a probe sequence of a fluorescent label is added under the condition allowed by the test condition, amplification is carried out on a GENIE II constant temperature amplification instrument, and real-time monitoring of nucleic acid amplification can be realized.
Drawings
FIG. 1 shows the specificity of real-time fluorescent RPA detection of EV; wherein, well 1 is EV; well 2-well 8 are MHV, MPV, SV, Reo-3, MVM, LCMV, and blank controls, respectively.
FIG. 2 shows the specificity of PCR detection of EV; wherein M is DNA Marker DL 2000; the virus samples detected in lanes 1-8 are: EV, 2.MHV, 3.MPV, 4.SV, 5.Reo-3, 6.MVM, 7.LCMV, 8. blank control.
FIG. 3 is a graph showing the sensitivity of real-time fluorescent RPA detection of EV; wherein the amplification curves are 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg and 0.1fg of the varicella virus DNA template from left to right respectively.
FIG. 4 shows a sensitivity test for detecting EV by RT-PCR; wherein M is DNA Marker DL 2000; the cDNA template concentrations in lanes 1-8 were 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg and, 0.1fg, respectively.
FIG. 5 fluorescent RPA detection of a mouse pox positive tissue sample; wherein, the 7 positive amplification curves are respectively liver, spleen, lung, kidney, heart, blood and intestine from top to bottom, and the non-amplification curve is a blank control.
FIG. 6 PCR detection of a mouse pox positive tissue sample; wherein M is DNA Marker DL 2000; the remaining lanes are: 1. blood, 2. liver, 3. spleen, 4. lung, 5. kidney, 6. heart, 7. intestine, 8. blank control.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1 design of real-time fluorescent PRA primers and probes
The conserved sequences of the varicella virus hemagglutinin protein-encoding genes isolated in different countries and regions were analyzed using Vector NTI Suite software, and RPA primers and probes were designed using Primer Express software, and the primers and probes were synthesized by Shanghai Rui Biotech Co.
TABLE 1 primer and Probe combinations
Wherein, the nucleotide sequence of the RPAY-EVP comprises the following components: SEQ ID NO.3+ FAM-dt + THF + BHQ1-dt + SEQ ID NO.4+ C3.
Example 2 establishment and optimization of the reaction System
1) Real-time fluorescent RPA detection
The reaction system is 50 μ L: 25. mu.L of 2-fold reaction buffer, 7.2. mu.L of 11mM dNTP, 5. mu.L of 10-fold probe enzyme mixture, 2.1. mu.L each of upstream and downstream primers (RPAY-EVF, RPAY-EVR) (10. mu.M each), and 0.6. mu.L of 10. mu.M fluorescent probe; after mixing evenly, 2.5 mul of 20 times core reaction solution and 1 mul of 50 times Exo reaction solution are added; after mixing, finally adding 2.5 mu L of 280mM magnesium acetate and 2 mu L of nucleic acid solution to be detected, wherein the reaction conditions are as follows: incubate at 39 ℃ for 25 min. After the reaction is finished, checking a fluorescence amplification curve and judging a result;
2) determination of optimal reaction time
To determine the optimal reaction time for RPA, 1X 103Copying/mu L of recombinant plasmid pT-EV-P72 DNA as a template, performing RPA amplification by respectively taking 10min, 20min, 25min, 30min, 35min and 40min as different reaction time, after the RPA reaction is finished, taking 5 mu L of product to perform 2% agarose gel electrophoresis, and analyzing an electrophoresis result by using an ultraviolet gel imaging system, wherein the result shows that when the RPA reaction time is 20min at 40 ℃, the electrophoresis can obtain a specific strip with the size of about 167bp, performing semi-quantitative analysis on the RPA product strip by using the ultraviolet gel imaging system, after the RPA reaction is performed for 30min, the strip is about 5.7 times of that when the strip is 20min, and the amplified strip after the reaction is 25min is not obviously increased, so that the optimal reaction time of the EV RPA is determined to be 25 min;
3) Determination of the optimum reaction temperature for RPA
In order to accurately compare the amplification efficiency of the RPA reaction at different temperatures, 50 mu L of RPA reaction system is prepared, after full mixing, amplification reactions are respectively carried out at 30, 35, 37, 39, 41 and 45 ℃, after 30min, 10 mu L of amplification liquid is respectively taken from each tube, and the result shows that the detection system can be effectively carried out at the temperature of 25-45 ℃, wherein the amplification efficiency is the best at 39 ℃, and the strip is the deepest during analysis by an ultraviolet gel imaging system, so the optimal reaction temperature is determined to be 39 ℃;
4) nucleic acid extraction and cDNA template preparation
For DNA virus samples, 100. mu.L of tissue supernatant or body fluid samples are taken, virus DNA extraction is carried out according to the instruction of a DNA extraction kit (commercially available, such as a kit from QIAGEN), and finally the DNA is dissolved in 50. mu.L of water to obtain a PCR template; for RNA virus samples, 100 mu L of supernatant or body fluid is taken, RNA is extracted by a TRIzol method, and then reverse transcription is carried out to obtain a cDNA template; the specific operation is as follows:
4.1 adding 700 mul of centrifuge tube of 65 ℃ preheating buffer solution 1 into 100 mul of tissue supernatant or body fluid sample, uniformly mixing, putting the centrifuge tube in 65 ℃ water bath for 20min, and reversing the centrifuge tube in the water bath process to mix the sample for a plurality of times;
4.2 adding 700 mu L chloroform, fully and uniformly mixing, and centrifuging at 12000 rpm for 5 min;
4.3 transferring the upper aqueous phase into a new centrifuge tube, adding 700 mu L of buffer solution 2, fully and uniformly mixing, transferring into an adsorption column, centrifuging at 12000 rpm for 30s, and discarding waste liquid;
4.4 adding 500 mul buffer solution 3 into the adsorption column, centrifuging at 12000 rpm for 30s, discarding the waste liquid, and putting the adsorption column into a collection tube;
4.5 adding 600 mul of rinsing liquid into the adsorption column, centrifuging at 12000 rpm for 30s, discarding the waste liquid, and putting the adsorption column into a collecting pipe; fully carrying out the operation once;
4.6 put the adsorption column into the collection tube, centrifuge at 12000 rpm for 2min, and discard the waste liquid. Placing the adsorption column at room temperature for several minutes;
4.7 transferring the adsorption column into another clean centrifugal tube, suspending and dripping 50-200 mu L of elution buffer solution into the middle part of the adsorption film, standing at room temperature for 3min, centrifuging at 12000 rpm for 2min, and collecting the solution into the centrifugal tube.
Example 3 PCR assay
The primers are designed aiming at the conserved region of the EV hemagglutinin protein coding gene, the size of an amplified gene fragment is 406bp, and the sequence of an upstream primer (EVF) thereof is as follows: 5 'ATA CGC TAC ACC TTA TCC TCA GAC AC 3', the sequence of the downstream primer (EVR) is: 5 'CGG TGA ATG TGT AGA TCC AGA TAG TA 3', adding 1 mu L of cDNA or DNA template, 2.5 mu L, dNTP 0.5.5 mu L of Taq enzyme concentrated buffer solution with the concentration being 10 times that of the PCR thin-wall tube, 0.25 mu L, Taq mu L of enzyme respectively for an upstream primer and a downstream primer, adding water to make up the total volume to 25 mu L, putting the PCR tube on a PCR instrument, and amplifying according to the following procedures: firstly, 94 ℃ for 3 min; then, 35 cycles of 94 ℃ for 15s, 56 ℃ for 15s, and 72 ℃ for 30s are carried out; finally 3min at 72 ℃. The PCR products were analyzed by a gel imaging system after agarose gel electrophoresis.
Example 4 specific assay for real-time fluorescent RPA detection of EV
The invention analyzes the conserved region gene sequence of the EV hemagglutinin encoding gene, designs a primer and a probe, establishes a real-time fluorescence RPA detection EV method, and respectively uses other viruses of mice, such as Mouse Hepatitis Virus (MHV), Mouse Parvovirus (MPV), mouse Sendai Virus (SV), mouse reovirus type III (Reo-3), mouse parvovirus (MVM) and lymphocyte choriomeningitis virus (LCMV) as a control, and simultaneously compares with a PCR method to confirm the specificity of the established method, and the result shows that the fluorescence signal of the EV positive sample is obviously enhanced after the reaction is 5-10 min through the fluorescence RPA detection, and the fluorescence signal is not detected in the control virus and bacteria samples (as shown in figure 1); only EV positive samples were specifically amplified by PCR detection, and all other virus controls were negative (as shown in figure 2).
Example 5 sensitivity assay for real-time fluorescent RPA detection of EV
In order to test the sensitivity of the established fluorescent RPA method, the DNA of the varicella virus is quantified and diluted in a gradient manner to prepare templates with the concentrations of 1 ng/muL, 100 pg/muL, 10 pg/muL, 1 pg/muL, 100 fg/muL, 10 fg/muL, 1 fg/muL and 0.1 fg/muL respectively, and then the fluorescent RPA method and the PCR method are established for comparative detection, so that the lower limit of the EV positive DNA template of the PCR detection sample is 1-10 pg/muL (shown in figure 5), and the lower limit of the EV DNA template of the RPA detection sample is also 1-10 pg/muL (shown in figure 4).
EXAMPLE 6 kit Assembly
The preparation of the kit components is completed according to two parts, namely extraction of nucleic acid and RPA reaction, and the data and the reagent components provided below are reagents required by single detection:
1) nucleic acid extraction (Room temperature storage)
The nucleic acid extraction reagent comprises a buffer solution 1(40ml), a buffer solution 2(40ml), a buffer solution 3(15ml), a rinsing solution (15ml), an elution buffer solution (15ml), an adsorption column and a collection tube;
2) RPA detection (-20 ℃ C. storage)
Primer probe mixed liquid tube: 2.1. mu.L of each 10. mu.M upstream and downstream primer, and 0.6. mu.L of 10. mu.M probe, for a total of 4.8. mu.L;
a reaction liquid pipe: 2 Xreaction buffer 25. mu. L, dNTP 8.2.2. mu.L, 10 Xprobe enzyme mixture 5. mu.L, 20 Xcore reaction solution 2.5. mu.L, 50 XExo reaction solution 1. mu.L; 2.5. mu.L of 280mM magnesium acetate.
Example 7 application of the kit-repetitive assay for real-time fluorescent RPA detection of EV
In order to further verify the reliability of the established nucleic acid detection method, positive samples of different tissue organs of the mouse pox are respectively detected by using fluorescent RPA and PCR, and the results show that the positive results are obtained by using the fluorescent RPA method and the PCR method to detect the tissue organs of heart, liver, spleen, lung, kidney, intestine, blood and the like of a sick mouse, and the coincidence rate of the positive results with the actual samples is 100 percent (as shown in figure 5 and figure 6).
Sequence listing
<110> Shanghai entry and exit inspection and quarantine bureau animal and plant and food inspection and quarantine technology center, Shanghai experiment animal and plant
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Claims (10)
1. A kit for detecting a varicella virus nucleic acid comprises an RPA reaction system and is characterized in that the RPA reaction system comprises an RPA primer probe mixed solution, the RPA primer probe mixed solution comprises a primer pair and a probe, the nucleotide sequence of the primer pair is shown as SEQ ID NO.1 and SEQ ID NO.2, and the probe is shown as SEQ ID NO.3+ FAM-dt + THF + BHQ1-dt + SEQ ID NO.4+ C3.
2. The kit of claim 1, wherein the concentration of the primers in the RPA reaction system is 5-20 μ M, preferably 10 μ M; the concentration of the probe is 5-10 μ M, preferably 10 μ M.
3. The kit of claim 2, wherein the RPA reaction system further comprises a core reaction solution, a reaction buffer, an Exo reaction solution, a probe enzyme mixture, magnesium acetate and dntps; preferably: the concentration of the magnesium acetate is 260-300 mu M, preferably 280 mu M;
and/or the concentration of the dNTP is 20-220 mu M, preferably 200 mu M.
4. The kit according to claim 3, wherein the primer-probe mixture comprises the primer pair and the probe, wherein the amount of each primer in the primer pair is 2.1. mu.L at a concentration of 10. mu.M, and the amount of the probe is 0.6. mu.L at a concentration of 10. mu.M; the amount of the core reaction solution is 2.5. mu.L, the amount of the reaction buffer solution is 25. mu.L, the amount of the probe enzyme mixture is 5. mu.L, and the amount of the Exo reaction solution is 1. mu.L; the amount of the magnesium acetate is 2.5 mu L, and the concentration is 280 mu M; the amount of the dNTP is 7.2 mu L, and the concentration is 11 mM; preferably: the core reaction solution is 20 times of core reaction solution;
and/or the reaction buffer solution is 2 times of the reaction buffer solution;
and/or the probe enzyme mixture is 10 times of the probe enzyme mixture;
and/or the Exo reaction solution is 50 times of the Exo reaction solution.
5. The kit of any one of claims 1 to 4, further comprising a negative control, preferably ddH2O。
6. A primer pair for detecting a varicella virus nucleic acid is characterized in that the nucleotide sequences of the primer pair are respectively shown as sequences SEQ ID NO.1 and SEQ ID NO. 2; preferably, the detection is RPA detection.
7. A probe for detecting a mousepox virus nucleic acid is characterized in that the probe is shown as SEQ ID NO.3+ FAM-dt + THF + BHQ1-dt + SEQ ID NO.4+ C3; preferably, the detection is RPA detection.
8. A combination for detecting a varicella virus nucleic acid comprises a primer pair and a probe, and is characterized in that the nucleotide sequences of the primer pair are respectively shown as sequences SEQ ID NO.1 and SEQ ID NO. 2; the probe is shown in SEQ ID NO.3+ FAM-dt + THF + BHQ1-dt + SEQ ID NO.4+ C3; preferably, the detection is RPA detection.
9. Use of a primer pair according to claim 6 and/or a probe according to claim 7, or a combination according to claim 8, for the preparation of a kit for the detection of a varicella virus.
10. A method for detecting a varicella virus for non-diagnostic purposes, comprising the steps of:
(1) extracting total DNA in a sample to be detected by using a DNA extraction reagent;
(2) performing an RPA reaction by using the total DNA extracted in the step (1) as a template and using an RPA reaction system in the kit according to any one of claims 1-5, wherein the time of the amplification reaction in the RPA reaction is preferably 25min, and the temperature is preferably 39 ℃;
(3) and analyzing the detection result.
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| CN113583862A (en) * | 2021-08-09 | 2021-11-02 | 上海实验动物研究中心 | Pathogen on-site nucleic acid detection device and application method |
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