CN110982935A - LAMP primer composition for detecting porcine delta coronavirus by adopting microfluidic chip technology and kit thereof - Google Patents
LAMP primer composition for detecting porcine delta coronavirus by adopting microfluidic chip technology and kit thereof Download PDFInfo
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Abstract
The invention belongs to the field of nucleic acid detection and diagnosis, and particularly relates to an LAMP primer composition for detecting porcine delta coronavirus by adopting a microfluidic chip technology and a kit thereof, aiming at solving the defects of time consumption, labor waste and high cost of the detection of the porcine delta coronavirus, the LAMP primer is designed according to a relatively conserved N gene of PDCoV, and the LAMP primer composition for detecting the porcine delta coronavirus is obtained after screening; on the basis, the invention also provides a kit combining the microfluidic chip technology and the LAMP technology and a detection method thereof, which are applied to the detection of the porcine delta coronavirus and can realize the rapid and instant detection of the porcine delta coronavirus, thereby solving the defects of time consumption, labor waste and high cost of the detection of the porcine delta coronavirus, improving the detection sensitivity and specificity, reducing the cost of labor and equipment and shortening the detection period.
Description
Technical Field
The invention belongs to the field of nucleic acid detection and diagnosis, and particularly relates to a LAMP primer composition for detecting porcine delta coronavirus by adopting a microfluidic chip technology and a kit thereof.
Background
Porcine delta coronavirus (PDCoV), also known as Porcine delta coronavirus or Porcine delta coronavirus, is a novel Porcine enterocoronavirus, and can cause diarrhea in pigs. The PDCoV virus is mainly transmitted through a digestive tract, the disease is fast, the transmission speed is high, pigs of various varieties and growth stages are susceptible to diseases, the main clinical symptoms are watery diarrhea, vomiting, dehydration and the like, and the death rate of the pigs is increased due to the mixed infection of the PDCoV virus, the TGEV (porcine transmissible gastroenteritis virus), the RV (porcine rotavirus) and the like. In 2009 WOO et al first detected PDCoV in swinery in hong kong, china, and thereafter appeared and reported in the united states, canada, thailand, korea, and other countries in succession. In recent years, the incidence of PDCoV has increased, causing severe economic losses to the swine industry.
PDCoV belongs to the family coronaviridae, the genus delta coronavirus, is an nonsegmented single-stranded positive-strand RNA virus, has a genome with a total length of about 25.4kb, includes a 5 'non-coding region and a 3' non-coding region, and encodes 4 major structural proteins, namely, a surface spike protein (S), a small membrane protein (E), a membrane protein (M), and a nucleocapsid protein (N). The protein has high N protein content, strong immunogenicity and good gene conservation, and is suitable for serving as a target gene for molecular detection. Clinical symptoms and pathological changes caused by various diarrhea viruses are similar, so that clinical diagnosis is difficult, and the diagnosis of PDCoV needs to be carried out by means of a laboratory molecular biological method. At present, the diagnostic methods applied to PDCoV are mainly: reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), fluorescent quantitative PCR, immunofluorescence, immunoelectron microscopy, indirect hemagglutination assay, and the like. The detection means has advantages of specificity and sensitivity, but the implementation of the detection methods needs professional personnel to operate, and the detection equipment is expensive and has long detection period, so the technical requirement is relatively high, and the detection method cannot be popularized and used. In addition, Point of care testing (POCT) can be used to perform on-site rapid diagnosis of diseases by means of small desktop devices or reagents, and has the advantages of rapidness, convenience, low testing cost, and the like.
The microfluidic POCT detection technology is a rapid detection technology developed in the last decade, and a microfluidic chip is also called a Lab-on-a-chip (LOC) and is a main platform for realizing the microfluidic technology. The microfluidic chip integrates the whole processes of sample preparation, reagent sample adding, sample separation, reaction progress and result detection in a conventional laboratory into a chip with a few square centimeters, realizes multi-target detection and analysis through a plurality of independent reaction chambers in the chip, has the advantages of portability, automation, integration, low cost, high flux, simple operation and the like, and provides wide prospects for the application of the microfluidic chip technology in various fields such as medical detection, disease diagnosis, food safety, environmental monitoring and the like.
The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method and is characterized in that 4 or 6 specific primers are designed for 6 regions of a target gene, and active strand displacement DNA polymerase is utilized to carry out strand displacement amplification reaction on a DNA template under the constant temperature condition (60-65 ℃). The method has the advantages that the reaction is carried out under the isothermal condition, and a complex temperature change procedure is not needed; high amplification efficiency, up to 10 in 1h10Carrying out amplification; the sensitivity is high, and is 2-5 orders of magnitude higher than that of the traditional PCR. At present, many researches combine loop-mediated isothermal amplification with a microfluidic chip so as to rapidly detect pathogens, drug-resistant gene loci, tumor marker genes and the like. For example, patent document (CN201820112618.0) discloses an analysis device for a thermostatted microfluidic chip based on microfluidicsThe chip control technology combines the LAMP constant temperature amplification technology, adopts the fluorescence detection technology, integrates heating, centrifugation, constant temperature amplification, reaction, fluorescence detection and curve result display into one device, judges the negative and positive according to the visual result, and realizes the instant detection.
In conclusion, the microfluidic chip technology and the LAMP technology are combined, a specific, stable, sensitive, rapid and efficient instant detection system can be established, the application value in the detection aspect of the porcine delta coronavirus is important, and the defects of time consumption, labor waste and high cost in the detection of the porcine delta coronavirus are expected to be overcome.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide the LAMP primer composition for detecting the porcine delta coronavirus by adopting a microfluidic chip technology.
The second purpose of the invention is to provide the application of the LAMP primer composition for detecting the porcine delta coronavirus by adopting the microfluidic chip technology in the preparation of the porcine delta coronavirus detection product.
The third purpose of the invention is to provide the LAMP kit for detecting the porcine delta coronavirus by adopting the microfluidic chip technology.
The fourth purpose of the invention is to provide the application of the LAMP kit for detecting the porcine delta coronavirus by adopting the microfluidic chip technology in the preparation of the porcine delta coronavirus detection product.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides an LAMP primer composition for detecting porcine delta coronavirus by adopting a microfluidic chip technology, which comprises an outer primer F3/B3, an inner primer FIP/BIP and a loop primer LB, wherein F3 has a sequence shown in SEQ ID NO: 1, B3 has the nucleotide sequence shown in SEQ ID NO: 2, FIP has the nucleotide sequence shown in SEQ ID NO: 3, BIP has the nucleotide sequence shown in SEQ ID NO: 4, LB has the nucleotide sequence shown in SEQ ID NO: 5.
The invention searches and obtains the full-length genome sequences of all PDCoV from a Genbank database, carries out homology analysis through BLAST software to find a target gene (N gene) sequence of PDCoV genome which is relatively conservative, designs LAMP primers for PDCoV detection by utilizing PrimeExplorer V5 software according to the N gene sequence, obtains 3 pairs of specific and sensitive primer compositions required by a microfluidic chip after screening, and is expected to provide an instant detection method combining the microfluidic chip technology and the LAMP amplification technology for the detection of the porcine Deltay coronavirus.
The invention also provides application of the LAMP primer composition for detecting the porcine delta coronavirus by adopting the microfluidic chip technology in preparation of a porcine delta coronavirus detection product.
The invention also provides an LAMP kit for detecting the porcine delta coronavirus by adopting a microfluidic chip technology, and the kit comprises the LAMP primer composition.
Preferably, in the LAMP primer composition, the molar ratio of the outer primer F3/B3, the inner primer FIP/BIP and the loop primer LB is 1:8:4, and the concentration of each primer component in the LAMP primer composition is 50 μ M.
Preferably, the kit further comprises a microfluidic chip, a constant temperature amplification premix, a reverse transcriptase solution and an RNase inhibitor, and of course, in order to improve the application range of the kit, other reagents which can be applied to the detection of the porcine delta coronavirus can also be included in the invention.
Preferably, in order to improve the LAMP amplification effect, the constant-temperature amplification premix solution is a reaction solution containing 800U/mL Bst DNA polymerase and 50 mu M fluorescent dye SYBR Green.
Preferably, in order to improve the LAMP amplification effect, the reverse transcriptase solution is M-MLV reverse transcriptase, and the RNase inhibitor is an RNase inhibitor; more preferably, the reverse transcriptase is M-MLV reverse transcriptase with a concentration of 5000U/mL, and the RNase inhibitor is RNase inhibitor with a concentration of 40U/. mu.L.
Preferably, in order to enlarge the detection capability of the microfluidic chip, the microfluidic chip comprises 8 reaction detection areas, each reaction detection area comprises a sample injection pool, a distribution pool, a capillary micro valve and an amplification pool which are sequentially communicated, and each reaction detection area is provided with 4 amplification pools.
The invention also provides application of the LAMP kit for detecting the porcine delta coronavirus by adopting the microfluidic chip technology in preparation of a porcine delta coronavirus detection product. Specifically, the method for detecting the porcine delta coronavirus by using the LAMP kit for detecting the porcine delta coronavirus by adopting the microfluidic chip technology comprises the following steps of:
s1, nucleic acid extraction of a sample to be detected: extracting nucleic acid of a diarrhea pig sample infected with porcine delta coronavirus;
s2, coating of LAMP primer composition: adding the LAMP primer compositions into corresponding amplification pools respectively, setting negative controls, and coating the LAMP primer compositions in the amplification pools after heating, drying, tabletting and film sealing and punching vacuum;
s3, LAMP reaction: mixing the constant-temperature amplification premix solution, a reverse transcriptase solution, an RNase inhibitor and nucleic acid of a sample to be detected, transferring the mixture into a sample injection pool of a microfluidic chip coated with an LAMP primer composition, sealing a membrane, and putting the membrane into a detection device for LAMP reaction;
and S4, judging whether the sample to be detected is a positive sample of the porcine delta coronavirus according to the condition of the fluorescence amplification curve after reaction, and if the amplification curve corresponding to the sample to be detected is S-shaped and the negative control has no amplification curve, judging that the sample to be detected is the positive sample of the porcine delta coronavirus.
Preferably, the LAMP primer composition coating method specifically comprises the following steps: mixing each primer component in the LAMP primer composition with a sucrose solution respectively to prepare a corresponding mixed solution, enabling the molar concentration of each primer component to be 0.15 mu M and the mass concentration of sucrose to be 1.0%, then adding the mixed solution into an amplification pool of a microfluidic chip, and coating the LAMP primer composition in the amplification pool after heating, drying, tabletting and sealing and punching vacuum.
Compared with the prior art, the invention has the beneficial effects that:
the invention designs LAMP primers on relatively conservative N genes by comparing all sequences of PDCoV in Genbank, and obtains a product after screeningThe LAMP primer composition is used for detecting the porcine delta coronavirus. Meanwhile, on the basis of the LAMP primer composition, the invention also provides a kit combining the microfluidic chip technology and the LAMP amplification technology, and the kit is used for establishing a rapid, sensitive, high-accuracy and strong-repeatability detection method for the porcine delta coronavirus; tests on specificity, sensitivity, repeatability and stability show that the detection method combining the microfluidic chip technology with the LAMP technology has high specificity, does not have cross reaction with other diarrhea pathogens of pigs, and has the lowest detection limit of 102The copies/mu L has better repeatability and stability, and can realize the rapid and instant detection of the porcine delta coronavirus when being applied to the detection of the porcine delta coronavirus, thereby solving the defects of time consumption, labor waste and high cost of the detection of the porcine delta coronavirus, improving the detection sensitivity and specificity, reducing the cost of labor and equipment and shortening the detection period; the rapid detection technology provided by the method can be popularized and applied to epidemiological investigation and epidemic situation monitoring of the porcine delta coronavirus, and has good practical significance and wide market prospect.
Drawings
FIG. 1 is a schematic structural diagram of a microfluidic chip used in an embodiment of the present invention;
the labels in the figure are: 1-reaction detection zone; 2-sample adding pool; 3-an amplification pool; 4-capillary microvalves; 5-a distribution pool;
FIG. 2 is a schematic diagram of the LAMP primer composition used in the embodiment of the present invention coated in a microfluidic chip;
the labels in the figure are: a-a sample detection well; b-negative control wells;
FIG. 3 shows the specific test result of detecting porcine delta coronavirus by combining the microfluidic chip technology with the LAMP technology;
FIG. 4 shows the sensitivity test result of the microfluidic chip technology combined with LAMP technology for detecting porcine delta coronavirus;
FIG. 5 shows the result of the test of the repeatability and stability of the detection of porcine delta coronavirus by the microfluidic chip technology in combination with LAMP technology.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 preparation of LAMP primer composition for detecting porcine delta coronavirus by microfluidic chip technology
The LAMP primer composition comprises the following design and synthesis steps:
firstly, retrieving a full-length genome sequence of all PDCoVs from a Genbank database, carrying out homology analysis through BLAST software to find a target gene (N gene) sequence of the PDCoV genome which is relatively conserved, designing an LAMP primer composition by using PrimeExplorer V5 software according to the obtained target gene sequence, and carrying out primer synthesis;
and then screening the synthesized primers, namely, screening the primers after dissolving the synthesized primers to finally obtain the specific and sensitive primer composition required by the microfluidic chip. The primer composition consists of an outer primer F3/B3, an inner primer FIP/BIP and a loop primer LB, wherein F3 has the sequence shown in SEQ ID NO: 1, B3 has the nucleotide sequence shown in SEQ id no: 2, FIP has the nucleotide sequence shown in SEQ ID NO: 3, BIP has the nucleotide sequence shown in SEQ ID NO: 4, LB has the nucleotide sequence shown in SEQ ID NO: 5. The nucleotide sequences of the outer primer F3/B3, the inner primer FIP/BIP and the loop primer LB are shown below:
F3：ACCACTCGTGTTACTTGGGT；
B3：ACGCTCCTGAGGTCTTCC；
FIP：TTGTTGGGGTTGCGTTTGGC-TAAGGGTTCGGGAGCTGAC；
BIP：GCTGCTACCTCTCCGATTCCC-TCTAGCGTTGAAGGGGTCA；
LB：GAGATGGCCCAGCTCAAGGT。
EXAMPLE 2 Structure of microfluidic chip
As shown in fig. 1, the micro-fluidic chip adopted by the present invention is a disc-shaped micro-fluidic chip, which is manufactured by shanghai rapid-invasive diagnostic products limited, and has a model number of 8 × 4, and comprises 8 reaction detection areas 1, each reaction detection area 1 comprises a sample introduction pool 2, a distribution pool 5, a capillary micro-valve 4 and an amplification pool 3 which are sequentially communicated, the sample introduction pool 2 is communicated with the distribution pool 5 through an arc-shaped channel, a sample introduction hole is arranged in the sample introduction pool 2, and the distribution pool 5 is communicated with a waste liquid pool and an exhaust hole through an arc-shaped channel; each reaction detection zone 1 is provided with 4 amplification cells 3.
Wherein, the sample injection pool 2 has the main function of loading reaction liquid; the distribution pool 5 is mainly used for uniformly distributing the reaction liquid to the amplification pool 3; the main function of the amplification pool 3 is to perform LAMP reaction; the capillary micro valve 4 mainly utilizes the retardation of the capillary to the liquid to control the movement of the fluid in the micro-fluidic chip.
Example 3 establishment of kit for detecting porcine delta coronavirus by microfluidic chip technology
The kit comprises the LAMP primer composition for detecting the porcine delta coronavirus in the embodiment 1 and the microfluidic chip in the embodiment 2, and further comprises a constant-temperature amplification premix, a reverse transcriptase solution and an RNase inhibitor.
In the LAMP primer composition for detecting the porcine delta coronavirus, the molar ratio of the outer primer F3/B3, the inner primer FIP/BIP and the loop primer LB is 1:8:4, and the concentration of each primer component in the LAMP primer composition is 50 mu M.
The isothermal amplification premix was a reaction solution containing 800U/mL of Bst DNA polymerase and 50. mu.M of fluorescent dye SYBRGreen (available from Shanghai Rapid diagnostic products, Ltd.).
The reverse transcriptase solution has a solvent of water and a solute concentration of 5000U/mL.
The RNase inhibitor has a solvent of water and a solute concentration of 40U/. mu.L.
Example 4 preparation of Positive Standard for porcine Deltay coronavirus LAMP detection method
RNA is extracted from a PDCoV positive disease sample, the RNA is reversely transcribed into cDNA, the outer primer F3/B3 obtained by screening in the embodiment 1 is used for PCR amplification to amplify PDCoV-N gene, and an amplification product with the same size as a target fragment is obtained. After the PCR product is recovered, the pGEM-T-PDCoV plasmid is linearized and purified by connecting with a pGEM-T easy vector, transforming and amplifying a recombinant plasmid containing a target fragment, identifying by PCR of bacterial liquid and sequencing, and then carrying out in vitro transcription according to a kit RibomaxTMThe instructions of the LargeScale RNA Production Systems (Promega) were used to transcribe the target gene to obtain a plasmid (TPDCoV-N-RNA) as a positive standard for LAMP detection of porcine delta coronavirus, the concentration of the plasmid being 1X 109copies/μL。
Example 5 methods of Using the kit described in example 3 to detect porcine delta coronavirus
(1) Extracting nucleic acid of a sample to be detected: nucleic acid from a sample of diarrhea pigs infected with porcine delta coronavirus was extracted according to the Magnetic Viral DNA/RNA Kit nucleic acid extraction Kit instructions from TIANGEN corporation.
(2) Coating of LAMP primer composition: as shown in fig. 2, each primer component in the LAMP primer composition for detecting porcine delta coronavirus is mixed with a sucrose solution to prepare a corresponding mixed solution, and the final concentration of each primer component and sucrose in the mixed solution is 0.15 μ M (molar concentration) and 1.0% (mass percentage), respectively; adding 1.5 mu L of mixed solution into corresponding amplification pools 3 of the microfluidic chip, arranging 2 PDCoV detection pools and 2 negative control pools in each reaction detection area 1, placing the microfluidic chip in a drying table at 60 ℃ for drying, tabletting and sealing a membrane, and coating the LAMP primer composition in each amplification pool 3 after punching and vacuum.
(3) LAMP reaction: mixing 10 mu L of constant-temperature amplification premix, 0.1 mu L of reverse transcriptase solution, 0.1 mu L of RNase inhibitor and 14.8 mu L of extracted nucleic acid of a sample to be detected, transferring the mixture to a sample injection pool 2 of a micro-fluidic chip coated with a primer composition, sealing a membrane in the sample injection pool 2, placing the prepared micro-fluidic chip in a centrifuge (Shanghai Sudoku diagnosis product Co., Ltd., type IGene techno TM SC-MA2000) with a fluorescence acquisition function, and instantly centrifuging at 1600rpm/min for 10s to uniformly mix reaction liquid; and centrifuging at 4600rpm/min for 30s to make the liquid flow into the amplification pool 3 under the action of centrifugal force, and reacting at 37 ℃ for 20 min. After the reaction is finished, according to the sequence displayed by the instrument program, the temperature is set to 63.5 ℃, the low-speed centrifugation rotating speed is 1600r/min, the low-speed centrifugation time is 10s, the high-speed centrifugation rotating speed is 4600r/min, the high-speed centrifugation time is 30s, and the reaction time is set to 60 min.
(4) And (4) interpretation of results: and (3) judging whether the sample to be detected is a sample with positive porcine delta coronavirus according to the condition of the fluorescent amplification curve corresponding to each amplification pool 3 displayed by the instrument after the reaction, if the fluorescent amplification curve corresponding to the sample to be detected is S-shaped on a display screen and the negative control has no amplification curve (namely the CT value is 0), judging that the sample to be detected is a sample with positive porcine delta coronavirus, otherwise, judging that the sample to be detected is a sample with negative porcine delta coronavirus.
Example 6 specificity assay for detection of porcine delta coronavirus by microfluidic chip in combination with LAMP
The method of embodiment 5 is adopted for detection, and the sample to be detected comprises porcine parvovirus, porcine encephalitis B virus, porcine circovirus type 2, hog cholera virus, porcine reproductive and respiratory syndrome virus, porcine acute diarrhea syndrome coronavirus, porcine delta coronavirus and negative control. The results of the detection are shown in FIG. 3. As can be seen from FIG. 3, the method for detecting porcine delta coronavirus by combining the microfluidic chip technology with the LAMP technology, which is established by the invention, does not have cross reaction with other porcine pathogens, and has good detection specificity.
Example 7 sensitivity test for detection of porcine delta coronavirus by microfluidic chip in combination with LAMP
The method of example 5 is adopted for detection, the PDCoV positive standard (TPDCoV-N-RNA) prepared in example 4 is diluted by 10 times of gradient and then used as a template for detection of microfluidic chip combined LAMP, and 7 gradients (l x 10) are totally obtained6～l×100copies/. mu.L) to determine their minimal detection limit. The results of the detection are shown in FIG. 4. According toFIG. 4 shows that the lowest detection limit of the detection method using the micro-fluidic chip technology combined with LAMP technology using TPDCoV-N-RNA standard as the template is 1X 102copies/μL。
Example 8 test for detecting the repeatability and stability of porcine delta coronavirus by combining microfluidic chip with LAMP
The method of example 5 is adopted for detection, the PDCoV positive standard (TPDCoV-N-RNA) prepared in example 4 is subjected to 10-fold gradient dilution and then is used as a template for microfluidic detection, and 3 dilutions are selected for testing (l x 10)6copies/μL，l×104copies/μL，l×102copies/. mu.L) were repeated 8 times per dilution, and the 3 dilutions of positive plasmid were tested for reproducibility and stability, the results are shown in FIG. 5. As can be seen from FIG. 5, the Coefficient of Variation (CV) within the group was determined for each dilution tested<5 percent, the method combines the microfluidic chip technology with the LAMP technology to detect the porcine delta coronavirus, and has better repeatability and stability.
The above examples show that the primer combination and the kit for detecting the porcine delta coronavirus by combining the microfluidic chip with the LAMP technology can quickly and accurately detect the porcine delta coronavirus, thereby overcoming the defects of time and labor waste of the existing porcine delta coronavirus detection technology, reducing the detection cost and labor intensity, improving the sensitivity and specificity and shortening the detection period. The method for judging the detection result by the amplification curve can meet the requirement of visualization, so that the method has the characteristics of rapidness and simplicity in operation in scientific research and production practice, and is suitable for on-site instant detection.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
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Claims (10)
1. The LAMP primer composition for detecting the porcine delta coronavirus by adopting a microfluidic chip technology is characterized by comprising an outer primer F3/B3, an inner primer FIP/BIP and a loop primer LB, wherein F3 has the nucleotide sequence shown in SEQ ID NO: 1, B3 has the nucleotide sequence shown in SEQ ID NO: 2, FIP has the nucleotide sequence shown in SEQ ID NO: 3, BIP has the nucleotide sequence shown in SEQ ID NO: 4, LB has the nucleotide sequence shown in SEQ ID NO: 5.
2. The application of the LAMP primer composition for detecting the porcine delta coronavirus by the microfluidic chip technology in the preparation of the porcine delta coronavirus detection product in claim 1.
3. An LAMP kit for detecting porcine delta coronavirus by adopting a microfluidic chip technology, which is characterized by comprising the LAMP primer composition of claim 1.
4. The LAMP kit for detecting the porcine deltacoronaviruses by adopting the microfluidic chip technology as claimed in claim 3, wherein in the LAMP primer composition, the molar ratio of the outer primer F3/B3, the inner primer FIP/BIP and the loop primer LB is 1:8: 4.
5. The LAMP kit for detecting the porcine delta coronavirus by adopting the microfluidic chip technology as claimed in claim 3, which is characterized by further comprising a microfluidic chip, a constant-temperature amplification premix, a reverse transcriptase solution and an RNase inhibitor.
6. The LAMP kit for detecting the porcine delta coronavirus by adopting the microfluidic chip technology as claimed in claim 5, wherein the microfluidic chip comprises 8 reaction detection areas (1), each reaction detection area (1) comprises a sample injection pool (2), a distribution pool (5), a capillary micro valve (4) and an amplification pool (3) which are sequentially communicated, and each reaction detection area (1) is provided with 4 amplification pools (3).
7. The LAMP kit for detecting the porcine deltacoronaviruses by adopting the microfluidic chip technology as claimed in claim 5, wherein the constant temperature amplification premixed solution is a reaction solution containing 800U/mL Bst DNA polymerase and 50 μ M fluorescent dye SYBRGreen.
8. The LAMP kit for detecting the porcine delta coronavirus by the microfluidic chip technology as claimed in claim 5, wherein the reverse transcriptase solution is M-MLV reverse transcriptase.
9. The LAMP kit for detecting the porcine deltacoronaviruses by adopting the microfluidic chip technology as claimed in claim 5, wherein the RNase inhibitor is an RNase inhibitor with a concentration of 40U/μ L.
10. The application of the LAMP kit of claims 3-9 in the preparation of porcine delta coronavirus detection products.
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| CN111394520A (en) * | 2020-04-20 | 2020-07-10 | 上海国际旅行卫生保健中心(上海海关口岸门诊部) | Primer group and detection kit for detecting new coronavirus based on RT-L AMP technology |
| CN111471802A (en) * | 2020-05-29 | 2020-07-31 | 华南农业大学 | Porcine delta coronavirus rapid detection primer, kit and application thereof |
| CN112342276A (en) * | 2020-11-09 | 2021-02-09 | 成都博奥独立医学实验室有限公司 | Method for detecting micro-fluidic chip by non-specific fluorescence adsorption of nucleic acid secondary structure |
| CN112522443A (en) * | 2020-12-18 | 2021-03-19 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Primer composition and kit for detecting porcine acute diarrhea syndrome coronavirus and application |
| CN113981140A (en) * | 2021-10-09 | 2022-01-28 | 广州达安基因股份有限公司 | Detection method of novel coronavirus delta mutant strain and nucleic acid detection kit |
| CN113981149A (en) * | 2021-11-23 | 2022-01-28 | 广东省农业科学院动物卫生研究所 | Porcine delta coronavirus detection primer group, probe, kit and application |
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| CN112342276A (en) * | 2020-11-09 | 2021-02-09 | 成都博奥独立医学实验室有限公司 | Method for detecting micro-fluidic chip by non-specific fluorescence adsorption of nucleic acid secondary structure |
| CN112522443A (en) * | 2020-12-18 | 2021-03-19 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Primer composition and kit for detecting porcine acute diarrhea syndrome coronavirus and application |
| CN113981140A (en) * | 2021-10-09 | 2022-01-28 | 广州达安基因股份有限公司 | Detection method of novel coronavirus delta mutant strain and nucleic acid detection kit |
| CN113981140B (en) * | 2021-10-09 | 2024-02-27 | 广州达安基因股份有限公司 | Novel coronavirus delta mutant strain detection method and nucleic acid detection kit |
| CN113981149A (en) * | 2021-11-23 | 2022-01-28 | 广东省农业科学院动物卫生研究所 | Porcine delta coronavirus detection primer group, probe, kit and application |
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