CN111270007A - Primer, micro-fluidic chip and system for detecting classical swine fever virus and application of primer, micro-fluidic chip and system - Google Patents
Primer, micro-fluidic chip and system for detecting classical swine fever virus and application of primer, micro-fluidic chip and system Download PDFInfo
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Abstract
A primer, a micro-fluidic chip, a system and application thereof for detecting classical swine fever virus are provided, wherein the primer is a primer group with the following sequences: p1: AGGGACTAGCCGTAGTGG, P2: AGGTCGTACCCCCATCAC, P3: GGTGAGCTTCTGCTCATGTCGA-AGCTCCCTGGGTGGTCTA, P4: CGAGATGCTATGTGGACGAGGG-TGTGAGTTCACCCTAGCGA. The kit thoroughly overcomes a plurality of defects of LAMP isothermal amplification, realizes the detection effects of simple and convenient operation, quick result output, multiple target spots, synchronization and integration, has the characteristics of high sensitivity and high specificity, and can realize the detection of a plurality of different indexes of the classical swine fever virus and the like of the same sample or the indexes of the classical swine fever virus of a plurality of samples for 1 time.
Description
Technical Field
The invention relates to the technical field of hog cholera virus detection, and particularly relates to a primer, a micro-fluidic chip and a system for detecting hog cholera virus and application thereof.
Background
Microfluidics (Microfluidics) refers to the science and technology involved in systems that process or manipulate tiny fluids using microchannels (tens to hundreds of microns in size), and is an emerging interdiscipline that involves chemical, fluid physics, microelectronics, new materials, biology, and biomedical engineering. Because of their miniaturization, integration, etc., microfluidic devices are commonly referred to as microfluidic chips, i.e., various basic operating units (preparation, reaction, separation, detection, etc.) are integrated on a chip of several square centimeters.
The current gene detection technology is based on PCR (polymerase chain reaction) technology and LAMP (loop-mediated isothermal amplification) technology. The PCR technology is more classical, parameters of a fluorescence quantitative PCR instrument are set after reaction liquid, primers and a template are well mixed, an on-machine test is carried out, Ct value is determined by collecting fluorescence and setting a threshold line, and whether a sample contains African swine fever virus or not is determined according to the Ct value. However, the reaction needs to be circulated in 2 different temperature areas, the requirement on instruments is high, the cost is relatively high, meanwhile, the PCR technology only needs 1 pair of amplification primers, the interference is easy, the specificity is relatively insufficient, the result generating time is long, the requirement on operation speciality is high, and the trace addition steps are multiple. The LAMP technology has 4 different specific primers, so the detection result accuracy is higher, but the isothermal reaction is adopted, a hot start enzyme similar to PCR is lacked, and nonspecific amplification is easily generated at the temperature rise stage of equipment, so the detection result is influenced.
At present, the swine fever epidemic disease is more prevalent, the living standard of people is seriously influenced, the living cost is increased, and the detection of the swine fever virus is particularly important for overcoming the treatment and the spread of the swine fever virus; the traditional detection methods of swine fever include virus isolation culture, immunofluorescence test, animal inoculation experiment, enzyme-linked immunosorbent assay (ELISA) and the like, and the methods have defects in the aspects of specificity, sensitivity, timeliness and the like, so that a high-sensitivity, high-specificity and rapid diagnosis method is very important in the detection of swine fever. The molecular biology detection technology comprises a reverse transcription-polymerase chain reaction (RT-PCR), a reverse transcription mediated isothermal amplification technology (RT-LAMP) and a fluorescent quantitative RT-PCR technology (Real-time RT-PCR), and the technologies have the characteristics of high specificity and high sensitivity, but the fluorescein labeled light of the fluorescent probe is extremely easy to decompose and needs to be stored away from light, the cost for synthesizing the fluorescent probe is high, the price of a fluorescent PCR instrument is high, and the like.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method which thoroughly overcomes the defects of LAMP isothermal amplification, realizes the detection effects of simple and convenient operation, quick result generation, multiple target spots, synchronization and integration, has the characteristics of high sensitivity and high specificity, and can realize the detection of multiple different indexes of classical swine fever virus and the like of the same sample or the indexes of classical swine fever virus of multiple samples for 1 time.
In order to solve the technical problems, the invention adopts the technical scheme that: a primer for detecting classical swine fever viruses comprises a primer group shown in the following sequence:
P1：AGGGACTAGCCGTAGTGG，
P2：AGGTCGTACCCCCATCAC，
P3：GGTGAGCTTCTGCTCATGTCGAAGCTCCCTGGGTGGTCTA，
P4：CGAGATGCTATGTGGACGAGGGTGTGAGTTCACCCTAGCGA。
the invention also provides a chip for detecting the swine fever virus, wherein the chip is fixed with the primer for detecting the swine fever virus and is a microfluidic chip amplified at constant temperature.
The invention also provides a swine fever virus detection system, which comprises the primer for detecting the swine fever virus and the chip.
The system of the invention can also comprise reagents and/or instruments and/or an amplification product data processor required for isothermal amplification, wherein the amplification product data processor can be used for distinguishing whether a product obtained by carrying out isothermal amplification on a sample to be detected by the nucleic acid isothermal amplification primer for detecting the swine fever virus contains a specific amplification product.
The instrument can be a constant-temperature amplification micro-fluidic chip nucleic acid analyzer, and the amplification product data processor is internally provided with software matched with the constant-temperature amplification micro-fluidic chip nucleic acid analyzer.
The reagent comprises reaction liquid, reverse transcriptase and an inhibitor; the target nucleic acid (sample to be tested) is added to the reagent.
Furthermore, the reagent also comprises an internal reference, and the performance of the reagent can be judged by adding the internal reference, and false negative in an experiment can be prevented.
Preferably, the reaction solution: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH4)2SO4, 8mM MgSO4, 0.1% Tween-20, 1.4mM dNTPs; bst enzyme, 800U/mL; 50uM SYBRGREEN fluorescent dye; the internal reference plasmid is 400 copies/ul; gold nanoparticles 4.0x10-6 mol/L, 0.6U (enzyme unit) FEN 1;
the reverse transcriptase is 5000U/mL;
the inhibitor is RNase, and the concentration of the inhibitor is 40U/. mu.L.
The concentrations are the concentrations of the components in the reagent.
Preferably, the microfluidic chip detection system according to the present invention, which comprises the above-described reagent, is:
the invention also provides a method for preparing any one of the following products 1) to 3), which also belongs to the protection scope of the invention, and the method comprises the following steps:
1) preparing four primer groups in the primer for detecting the hog cholera virus;
2) preparing the chip, wherein the chip contains a primer for detecting the hog cholera virus;
3) the system is prepared, and the system comprises the primer for detecting the classical swine fever virus in the step 1) and/or the chip in the step 2).
The invention also provides any one of the following applications of the primer for detecting the hog cholera virus, which also belongs to the protection scope of the invention:
1) the application in the preparation of the chip;
2) use in the preparation of said system;
3) the application of the reagent or the kit in the preparation of the hog cholera virus detection reagent or the kit.
The invention also provides a method for detecting whether a sample to be detected contains classical swine fever virus, which comprises the following steps:
a) searching a target sequence through NCBI GenBank, designing primers aiming at the target sequence, fixing the obtained primers at corresponding positions of the microfluidic chip respectively, and packaging the microfluidic chip;
b) mixing reaction liquid, reverse transcriptase, inhibitor and nucleic acid template extracted from pork and various organs, blood, excrement, environmental sample and cell culture, adding the mixture into the encapsulated microfluidic chip sample, allowing the mixture to enter a microfluidic chip reaction hole, and performing constant temperature amplification;
c) and if the sample contains the target fragment, performing isothermal amplification, effectively combining an amplification product with a fluorescent substance, capturing a fluorescent signal in real time by using a fluorescence detector, intuitively reacting the generation of the amplification product, and judging whether the sample contains Classical Swine Fever Virus (CSFV) according to the appearance time, intensity and position of the real-time fluorescent signal.
Preferably, the microfluidic chip in b) above is used for isothermal amplification, specifically: because the method adopts constant temperature amplification, the temperature-variable processes of denaturation, annealing, extension and the like of PCR amplification are not needed, the whole reaction process is finished under the constant temperature condition, and the amplification program comprises the following steps: the temperature was set at 63.5 ℃ and the reaction time was set at 30 min. And (3) running a program: the low-speed centrifugation rotating speed is 1600r/min, the low-speed centrifugation time is 10sec, the high-speed centrifugation rotating speed is 4600r/min, and the high-speed centrifugation time is 30 sec.
Preferably, after the mixing reaction in the step b), the mixture is added into a packaged microfluidic chip and then can be subjected to freeze-drying, namely, the reagent is pre-embedded and freeze-dried on the chip; the above-mentioned freeze-drying process is characterized by that the reaction liquor, reverse transcriptase and inhibitor can be embedded and freeze-dried.
The invention has the advantages and beneficial effects that:
1. the specific primer sequence set by the invention has the advantages of high detection stability and high accuracy.
2. Nucleic acid isothermal amplification microfluidic chip field operability: the reagents on the nucleic acid constant-temperature amplification chip are pre-embedded and freeze-dried (the reagents contain primers), and can be stored at normal temperature, and the micro-fluidic chip detection system can be provided with a large-capacity rechargeable lithium battery, so that the micro-fluidic chip detection system is convenient to use without an external power supply and is suitable for field quick detection.
3. The invention combines the micro-fluidic chip and the constant temperature gene detection technology, gives full play to the advantages of the micro-fluidic chip and the constant temperature gene detection technology, greatly accelerates the analysis speed (within half an hour), greatly improves the detection convenience (fool operation), and achieves the aims of quick and accurate detection and simple and convenient operation; the analysis sensitivity is greatly improved (<100copies), the biochemical reagents are greatly reduced compared with the traditional PCR technology (20% of the PCR technology), the use cost is greatly reduced, and the like. The research on the aspect fills the blank in China and is in the leading position internationally.
4. The gene detection technology mainly comprises a PCR (polymerase chain reaction) technology and an LAMP (loop-mediated isothermal amplification) technology at present, the PCR technology is more classical, but the reaction needs to circulate in 2 different temperature areas, the requirement on instruments is high, the cost is relatively high, and meanwhile, the PCR technology only needs 1 pair of amplification primers, so that the PCR technology is easy to interfere and has relatively insufficient specificity. The LAMP technology has 4 different specific primers, so the detection result accuracy is higher, but the isothermal reaction is adopted, a hot start enzyme similar to PCR is lacked, and nonspecific amplification is easily generated at the temperature rise stage of equipment, so the detection result is influenced. The invention takes the improved LAMP technology as the gene amplification reaction principle, and gold nanoparticles are added into the reagent, so that ssDNA and protease can be adsorbed, the nonspecific reaction in the heating process is inhibited, the purpose of hot start is achieved, and the nonspecific reaction in the heating process is avoided; simultaneously, FEN1 is added to act together with Bst enzyme, so that the double-stranded part of the stem-loop structure encountered in amplification is replaced and sheared, and finally, an amplification product is specifically formed; by combining the improved LAMP technology and the micro-fluidic chip technology, an accurate detection result can be rapidly given, and the purpose of joint detection of a plurality of different indexes of the same sample is achieved; meanwhile, the reaction reagent is embedded on the microfluidic chip, and a user only needs to add a sample, so that the operation is simple and convenient; the instrument can be provided with a lithium battery, and is convenient for quick field inspection. Compared with the LAMP tubular constant temperature technology on the market, the micro-fluidic chip solution provided by the intelligent movable nucleic acid micro-fluidic detector thoroughly overcomes the defects of LAMP isothermal amplification, and realizes the detection effect of simple operation, multiple targets, synchronization and integration.
Drawings
FIG. 1 is a process flow chart of the microfluidic detection of swine fever according to the present invention.
Detailed Description
The present invention will be described in further detail below by way of examples, but the present invention is not limited to only the following examples.
In the reagent pre-embedding step, an automatic spot spraying system of the American Biodot company is adopted, reagents (including primers) are sprayed in an amplification pool (the amplification pool is a reaction pool arranged on a chip), and for 2ul of trace spot sample, the error can be controlled within 2%.
The spotted chip needs to be attached with a bottom film, so that the amplification pool becomes a closed space, and meanwhile, the volume of the amplification pool is ensured to be 5ul, so as to ensure the consistency of a reaction system during detection. An automatic film pressing machine is adopted to automatically paste the base film on the chip, and the consistency of film pasting is guaranteed, so that the volume error of the chip reaction tank in batches and among batches is guaranteed to be in a controllable and allowable range to the maximum extent.
The chip of the application can realize that one-time detection can load 8 samples (8 samples), and the number of different detectable indexes of the chip is different.
Some of the components referred to in the present application, such as components in the reaction solution, are commercially available or those conventionally used and available in the industry, unless otherwise specified.
Examples
Searching a target sequence through NCBI GenBank, and designing a primer aiming at the target sequence, wherein the designed primer sequence is shown in the following table 1 (the corresponding sequence table is provided with sequence ordering):
TABLE 1 primer sequences
| P1 | AGGGACTAGCCGTAGTGG |
| P2 | AGGTCGTACCCCCATCAC |
| P3 | GGTGAGCTTCTGCTCATGTCGA-AGCTCCCTGGGTGGTCTA |
| P4 | CGAGATGCTATGTGGACGAGGG-TGTGAGTTCACCCTAGCGA |
And fixing the reaction liquid, the reverse transcriptase and the inhibitor at corresponding positions of the microfluidic chip respectively, packaging the microfluidic chip, mixing and reacting the reaction liquid, the reverse transcriptase and the inhibitor with a nucleic acid template extracted from pork and various visceral organs thereof, blood, excrement, an environmental sample and cell culture, adding the mixture into the packaged microfluidic chip, putting the packaged microfluidic chip into a microfluidic chip detector with a centrifugal function, a constant temperature function and real-time fluorescence detection, and driving the sample into a microfluidic chip reaction hole by utilizing centrifugal force to perform constant temperature amplification. And if the sample contains the target fragment, performing isothermal amplification, effectively combining an amplification product with a fluorescent substance, capturing a fluorescent signal in real time by using a fluorescence detector, intuitively reacting the generation of the amplification product, and judging whether the sample contains CSFV according to the appearance time, the intensity and the position of the real-time fluorescent signal.
The detection process of the microfluidic chip method is specifically shown in the attached figure 1.
(1) The detection system of the microfluidic chip is specifically shown in table 2:
TABLE 2 detection system for microfluidic chip
In the above table: reaction solution: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH)4)2SO4，8mM MgSO40.1% Tween-20, 1.4mM dNTPs; bst enzyme, 800U/mL; 50uM SYBRGREEN fluorescent dye; the internal reference plasmid is 400copies/ul or 0; the gold nanoparticles are 4.0x10-6 mol/L; 40U/. mu.L; 0.6U FEN 1;
reverse transcriptase, 5000U/mL;
RNase inhibitor, 40U/. mu.L.
(2) And (3) performing on-machine amplification of the microfluidic chip:
because the method adopts constant temperature amplification, the temperature-variable processes of denaturation, annealing, extension and the like of PCR amplification are not needed, the whole reaction process is finished under the constant temperature condition, and the amplification program comprises the following steps: the temperature was set at 63.5 ℃ and the reaction time was set at 30 min. And (3) running a program: the low-speed centrifugation rotating speed is 1600r/min, the low-speed centrifugation time is 10sec, the high-speed centrifugation rotating speed is 4600r/min, and the high-speed centrifugation time is 30 sec.
(3) And (3) judging the result of the microfluidic chip:
(3.1) setting of threshold line of micro-fluidic chip detector
The threshold line is set to 800 (which can be adjusted according to the actual situation, the setting principle is that the threshold line just exceeds the highest point of the atypical S-type amplification curve, and the Ct value is displayed as 30), and the instrument matching software automatically analyzes the result. The Ct value in this context is understood to be the time of the amplification reaction, as opposed to the Ct value in fluorescent quantitative PCR.
(3.2) quality control (when using internal reference)
When the Ct value of the internal reference is less than 30, an amplification curve appears, and the experimental result is effective.
The functions of internal reference: the effectiveness of the reagents was judged while preventing false negatives in the experiment.
The internal reference may be any commercially available internal reference that meets the requirements of the chip of the present application.
(3.3) determination of results
(3.3.1) conditions for establishing the experiment
Positive control: ct <30, and the reaction well of the positive control has a remarkable typical S-shaped amplification curve.
Negative control: ct <30, no amplification curve for reaction wells of negative control.
(3.3.2) determination criteria
Positive: the Ct of the reaction hole is less than 30, and the reaction hole has an obvious amplification curve and is judged to be positive.
Negative: the Ct of the reaction hole is less than 30, no amplification curve exists, and the reaction hole is judged to be negative.
Carrying out constant-temperature amplification on the microfluidic chip on a microfluidic chip detector, carrying out real-time fluorescence detection by the detector, judging and reading according to an effective amplification curve of the fluorescence detection, and judging that any hole or multiple holes are positive if a standard S-shaped amplification curve exists in the hole, namely the sample contains viral nucleic acid corresponding to the detection hole; wells without amplification curve were judged negative, i.e., the sample did not contain viral nucleic acid corresponding to the detection well.
(4) Verification of sensitivity and detection limits
(4.1) test materials
Reagent: reaction solution; 1X 106copies/μL、1×105copies/μL、1×104copies/μL、1×103copies/μL、1×102copies/μL、1×101A plasmid with African swine fever virus gene fragment of copies/mu L and 1 × 100 copies/mu L; and (5) negative control.
The instrument comprises the following steps: a constant temperature amplification instrument; a palm centrifuge; a pipette.
(4.2) the detection system is specifically shown in Table 3:
TABLE 3 detection System
And carrying out experimental operation according to the detection system, and then putting the same chip into a constant-temperature amplification instrument for experimental detection.
(4.3) results
TABLE 4 amplified Ct values of detection limits
From the results of the detection limits of Table 4 above, the lowest detection limit was 1X 102In the case of copies/. mu.L plasmid, the Ct value is about 13 min.
(5) Verification of repeatability
(5.1) test materials
Reagent: reaction solution; 1X 104Plasmid of copies/. mu.L; a hog cholera vaccine; and (5) negative control.
The instrument comprises the following steps: a constant temperature amplification instrument; a palm centrifuge; a pipette.
(5.2) detection System
1×104Plasmid detection system of copies/. mu.L was performed according to 4.4.1.2; the swine fever vaccine is carried out according to the method (4.1), and then the chip is put into a constant temperature amplification instrument for test detection.
(5.3) results
TABLE 5 amplification Ct values for repeated experiments
From the results of the repeated tests in table 5 above, CV% values < 4% are satisfactory.
(6) Verification of specificity
(6.1) test materials
Reagent: reaction solution; porcine reproductive and respiratory syndrome live vaccine nucleic acid; porcine pseudorabies live vaccine nucleic acid; porcine circovirus type 2 culture fluid nucleic acid; porcine parvovirus cell culture fluid nucleic acid and negative control.
The instrument comprises the following steps: a constant temperature amplification instrument; a palm centrifuge; a pipette.
(6.2) detection System
The experimental operation was carried out with reference to the detection system in the above 1, and then the chip was put into an isothermal amplification apparatus for experimental detection.
(6.3) results
TABLE 6 amplification Ct values for repeated experiments
According to the embodiment, the improved LAMP technology is used as the gene amplification reaction principle for the first time, the gold nanoparticles are added into the reaction system, the purpose of hot start is achieved, and the non-specific reaction in the temperature rise process is avoided; the improved LAMP technology and the improved microfluidic chip technology are combined, so that an accurate detection result can be rapidly given, the purpose of joint detection of a plurality of different indexes of the same sample is achieved, meanwhile, the reaction reagent is pre-embedded on the microfluidic chip, a user only needs to add the sample, the operation is simple and convenient, and the instrument is provided with a lithium battery, so that the on-site quick detection is facilitated.
Sequence listing
<110> Ningbo love Gene science and technology Co., Ltd
Primer, micro-fluidic chip and system for detecting hog cholera virus and application of primer, micro-fluidic chip and system
<130>2019
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>18
<212>DNA
<213> Artificial sequence ()
<400>1
agggactagc cgtagtgg 18
<210>2
<211>18
<212>DNA
<213> Artificial sequence ()
<400>2
aggtcgtacc cccatcac 18
<210>3
<211>40
<212>DNA
<213> Artificial sequence ()
<400>3
ggtgagcttc tgctcatgtc gaagctccct gggtggtcta 40
<210>4
<211>41
<212>DNA
<213> Artificial sequence ()
<400>4
cgagatgcta tgtggacgag ggtgtgagtt caccctagcg a 41
Claims (12)
1. A primer for detecting classical swine fever viruses is characterized in that: the primer is a primer group with the following sequences:
P1：AGGGACTAGCCGTAGTGG，
P2：AGGTCGTACCCCCATCAC，
P3：GGTGAGCTTCTGCTCATGTCGA-AGCTCCCTGGGTGGTCTA，
P4：CGAGATGCTATGTGGACGAGGG-TGTGAGTTCACCCTAGCGA。
2. a chip for detecting classical swine fever virus, wherein the primer for detecting classical swine fever virus according to claim 1 is immobilized on the chip.
3. The hog cholera virus detection chip of claim 2, wherein: the chip is a constant temperature amplification micro-fluidic chip.
4. A classical swine fever virus detection system, comprising the primer for detecting classical swine fever virus according to claim 1.
5. The hog cholera virus detection system of claim 4, comprising a chip according to claim 2 or 3.
6. The hog cholera virus detection system of claim 4 or claim 5, wherein: the system also comprises reagents and/or instruments required for isothermal amplification and/or an amplification product data processor, wherein the amplification product data processor is used for distinguishing whether a product obtained by carrying out isothermal amplification on a sample to be detected by the nucleic acid isothermal amplification primer for detecting the classical swine fever virus contains a specific amplification product.
7. The hog cholera virus detection system of claim 6, wherein: the reagent comprises reaction liquid, reverse transcriptase and an inhibitor; preferably, the composition also comprises an internal reference;
the reaction solution: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH)4)2SO4，8mM MgSO40.1% Tween-20, 1.4mM dNTPs; bst enzyme, 800U/mL; 50uM SYBRGREEN fluorescent dye; the internal reference plasmid is 400 copies/ul; gold nanoparticles 4.0x10-6mol/L；0.6U FEN1；
The reverse transcriptase is 5000U/mL;
the inhibitor is an RNase inhibitor and is 40U/. mu.L.
8. The hog cholera virus detection system of claim 7, wherein: the micro-fluidic chip detection system formed by the reagent is as follows:
9. the hog cholera virus detection system of claim 8, wherein: the instrument is a constant-temperature amplification microfluidic chip nucleic acid analyzer, and the amplification product data processor is internally provided with software matched with the constant-temperature amplification microfluidic chip nucleic acid analyzer.
10. A method for preparing any of the following products 1) -3), comprising the steps of:
1) preparing nucleic acid isothermal amplification primers for detecting classical swine fever virus according to claim 1;
2) preparing the chip of claim 2 or 3;
3) preparing a system as claimed in any one of claims 4 to 8.
11. The application of the nucleic acid isothermal amplification primer for detecting the classical swine fever virus comprises the following steps:
1) the application in the preparation of the chip;
2) use in the preparation of said system;
3) the application of the reagent or the kit in the preparation of the hog cholera virus detection reagent or the kit.
12. A method for detecting whether a sample to be detected contains classical swine fever virus or not is characterized by comprising the following steps:
searching a target sequence through NCBI GenBank, designing primers aiming at the target sequence, fixing the primers at corresponding positions of the microfluidic chip respectively, and packaging the microfluidic chip;
mixing reaction liquid, reverse transcriptase, an inhibitor and a nucleic acid template extracted from pork and various organs thereof, blood, excrement, an environmental sample and cell culture for reaction, adding the mixture into a packaged micro-fluidic chip, putting the micro-fluidic chip into a micro-fluidic chip detector with a centrifugal function, a constant temperature function and real-time fluorescence detection, and driving a sample to enter a micro-fluidic chip reaction hole by utilizing centrifugal force for constant temperature amplification;
if the sample contains the target fragment, the isothermal amplification is carried out, the amplification product is effectively combined with the fluorescent substance, the fluorescent signal is captured in real time by the fluorescent detector, the generation of the amplification product is intuitively reacted, and whether the sample contains the classical swine fever virus or not is judged according to the appearance time, the intensity and the position of the real-time fluorescent signal;
the amplification program of the constant temperature amplification of the microfluidic chip comprises the following steps: the temperature was set at 63.5 ℃ and the reaction time was set at 30 min. And (3) running a program: the low-speed centrifugation rotating speed is 1600r/min, the low-speed centrifugation time is 10sec, the high-speed centrifugation rotating speed is 4600r/min, and the high-speed centrifugation time is 30 sec.
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| CN112210624A (en) * | 2020-10-30 | 2021-01-12 | 浙江省农业科学院 | LAMP primer and kit for efficient triple detection of three pathogens of Chinese softshell turtles |
| CN112210624B (en) * | 2020-10-30 | 2023-07-25 | 浙江省农业科学院 | LAMP primer and kit for efficiently detecting three pathogens of trionyx sinensis in triple mode |
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