CN110885900A - Freeze-drying microchip, kit and method for identifying classical strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and strain NADC30-Like - Google Patents
Freeze-drying microchip, kit and method for identifying classical strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and strain NADC30-Like Download PDFInfo
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Abstract
The invention discloses a freeze-drying microchip, a kit and a method for identifying classical strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and a strain NADC30-Like, belonging to the field of molecular detection. The freeze-drying microchip is characterized in that: a fluorescent PCR reaction system for identifying classical strains of porcine reproductive and respiratory syndrome virus and an NADC30-Like strain is fixed on the microchip by freeze-drying; the fluorescent PCR reaction system comprises: the following primers and probes: an upstream primer of a classical strain of the porcine reproductive and respiratory syndrome virus, a downstream primer of a classical strain of the porcine reproductive and respiratory syndrome virus, and a Taqman probe sequence of the classical strain of the porcine reproductive and respiratory syndrome virus; the kit comprises an NADC30-Like strain upstream primer, an NADC30-Like strain downstream primer and an NADC30-Like strain Taqman probe. The detection kit can be used for simultaneously detecting the porcine reproductive and respiratory syndrome classical strain and the NADC30-Like strain viruses, and the detection method of the detection kit has high accuracy, specificity and sensitivity and short detection time.
Description
Technical Field
The invention relates to the technical field of virus molecular biological detection, in particular to a freeze-drying microchip, a kit and a method for identifying classical strains and an NADC30-Like strain of porcine reproductive and respiratory syndrome virus.
Background
Porcine Reproductive and Respiratory Syndrome (PRRS) is colloquially referred to as "Porcine reproductive and respiratory syndrome". The disease is an important epidemic disease of pigs caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) to cause reproductive and respiratory disorders. PRRSV mainly infects pigs, especially sows, which seriously affects their reproductive function, and is clinically characterized by abortion, stillbirth, mummy, weak fetus, dyspnea, and transient cyanosis of the skin of both ears during the disease process, so the disease is also called blue-ear disease. The disease is the first disease which harms pig farms in recent years, can cause abortion, stillbirth, abnormal oestrus and immunosuppression of sows, can also cause diarrhea and respiratory diseases of commercial pigs, and seriously influences the growth speed and health of the pigs. The classical porcine reproductive and respiratory syndrome strain has certain pathogenicity but lower virulence, and is the best choice for preventing and treating highly pathogenic porcine reproductive and respiratory syndrome as a live porcine reproductive and respiratory syndrome vaccine. The strain NADC30 is discovered in China in 2013 for the first time, then continuous variation is carried out, the strain is recombined with domestic porcine reproductive and respiratory syndrome virus strains and is named as NADC30-Like strain, and a few of the strain NADC30-Like strains have strong toxicity. National NADC from 2015, the scholars performed epidemiological investigations on outbreaks of the blue ear disease NADC30-Like strain. The experimental result shows that the strain of the porcine reproductive and respiratory syndrome NADC30-Like has been widely spread in China and can cause clinical diseases of pigs.
In recent years, although scientists have conducted extensive research on PRRSV, no effective method for eradicating PRRS has been found yet, which relies on timely and accurate diagnosis and control of the epidemic. The diagnosis is that the antigenicity difference of PRRSV strains with different genotypes and pathogenicity is large, the diagnosis technology and the prevention and control vaccine are different, and the diagnosis of different classifications according to the detection purpose is the first priority of the disease prevention and control. At present, the method adopted for detecting the porcine reproductive and respiratory syndrome virus mainly adopts the traditional virus separation identification, ELISA method and common PCR detection. The virus separation operation is complicated, the time consumption is long, and the omission factor is high; the ELISA method is relatively simple and rapid, but has low specificity and sensitivity for a sample with trace or more impurities, and can cause missed diagnosis or misdiagnosis. Since birth, PCR, a novel molecular biology technique, has high specificity and sensitivity, and can obtain a large number of target fragments through gene amplification in a short time, so that PCR becomes one of the important means for detecting PRRSV at present. However, the common PCR still has the disadvantages of complicated operation, long detection time, easy pollution and the like. Therefore, the market at present urgently needs reagent products which are rapid, accurate, high in sensitivity, portable and capable of being used for field operation detection and differential diagnosis.
Disclosure of Invention
Based on the above needs in the art, the present invention aims to provide a freeze-drying microchip and a kit for identifying porcine reproductive and respiratory syndrome virus classical strains and NADC30-Like strains, so that the microchip and the kit can rapidly and effectively detect porcine reproductive and respiratory syndrome virus, and have high accuracy, specificity and sensitivity and good repeatability.
In order to achieve the purpose, the invention adopts the following technical scheme:
a freeze-drying microchip for identifying classical strains of porcine reproductive and respiratory syndrome virus and an NADC30-Like strain, which is characterized in that a fluorescent PCR reaction system for identifying classical strains of porcine reproductive and respiratory syndrome virus and an NADC30-Like strain is fixed on the microchip by freeze-drying;
the fluorescent PCR reaction system comprises: the following primers and probes:
classical strain upstream primer of porcine reproductive and respiratory syndrome virus: 5 '-TGTTGTGACTTGAGCGTCGAT-3',
classical strain downstream primers for porcine reproductive and respiratory syndrome virus: 5 '-CAAAAGGCCAGGAACCGTAA-3',
classical strain Taqman probe sequence for porcine reproductive and respiratory syndrome virus: 5 '-TTTGTGATGCTCGTCAGG-3';
NADC30-Like strain upstream primer: 5 '-GCCTCGCTCAGAACTTCCT-3',
NADC 30-downstream primer of Like strain: 5 '-CACCACGATGTAGGCTTCAG-3',
NADC30-Like strain Taqman probe sequence: 5 '-AGCATCTCGTCTCC-3'.
The fluorescent PCR reaction system further comprises: taq enzyme, reverse transcriptase, trehalose, Tris-Cl, dNTP, Mg2+；
Preferably, the 5' end of the Taqman probe of classical strain and NADC30-Like strain of the porcine reproductive and respiratory syndrome virus is marked with a fluorescent reporter group; the 3' end is marked with a fluorescence quenching group.
The fluorescent PCR reaction system comprises: upstream primer 0.4. mu.M; downstream primer 0.4. mu.M; taqman probe 0.4. mu.M; DNA polymerase 0.5U/. mu.L; reverse transcriptase 0.5U/. mu.L; dNTP 0.3 mM; mg (magnesium)2+3 mM; 5 mu M of trehalose; Tris-Cl5mM, the balance of sterilized deionized water, the total volume of 36 mu L, and each pore volume of 1.2 mu L;
preferably, the fluorescence reporter group marked at the 5' end of the Taqman probe of classical strain of porcine reproductive and respiratory syndrome virus is a FAM fluorescence reporter group;
the fluorescence reporter group marked at the 5' end of the Taqman probe of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) NADC30-Like strain is a ROX fluorescence reporter group;
the fluorescence quenching group marked at the 3' end of the Taqman probe of classical strain and NADC30-Like strain of the porcine reproductive and respiratory syndrome virus is MGB quenching group.
The microchip is provided with a plurality of sample adding holes; the fluorescent PCR reaction system is fixed in the sample adding hole by freeze-drying;
preferably, the microchip on the sample hole is 30; the structure of the microchip is matched with the structure of a sample adding plate of the PCR instrument.
The freeze-drying comprises the following steps: freezing the microchip filled with the fluorescent PCR reaction system at-80 ℃ for 1h, and then carrying out equipment freeze-drying;
preferably, the device lyophilization comprises: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the pre-freezing time is kept for 1h, then the equipment is vacuumized, and the freeze drying is kept for 1 h; the drying stage was resolved, the spacer temperature was again raised to-25 ℃ for 1h, then raised to 37 ℃ and held for 2h, and finally lowered to 25 ℃ and held for 1 h.
A kit for identifying classical strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) from strain NADC30-Like, comprising said freeze-dried microchip.
The kit further comprises: the diluent, 10 Xdiluent, was diluted to 2 Xdiluent with nuclease-free water for dripping into the wells of the freeze-dried microchip, and the freeze-dried microchip was subjected to fluorescent PCR amplification on a fluorescent PCR instrument.
The kit further comprises: mineral oil, close the freeze-drying microchip on the sample hole;
positive control, specifically, a genomic cDNA mixture of classical porcine reproductive and respiratory syndrome virus strains and an NADC30-Like strain;
negative control, specifically nuclease-free water.
The method for identifying classical strains of porcine reproductive and respiratory syndrome virus and the strain NADC30-Like is characterized in that the freeze-drying microchip is adopted and/or the kit is used for carrying out fluorescence PCR amplification on a sample to be detected.
After a sample to be detected and diluent are added into a sample adding hole of the freeze-drying microchip, placing the freeze-drying microchip on a fluorescence PCR instrument for performing fluorescence PCR amplification;
preferably, the diluent is 10 × buffer;
further preferably, the reaction procedure of the fluorescent PCR amplification is: 10min at 50 ℃; 1min at 95 ℃; the fluorescence signal was detected at the end of extension in 40 cycles of 1 cycle of 95 ℃ 5s and 60 ℃ 15 s.
The invention provides a fluorescent RT-PCR detection kit, which comprises a freeze-drying microchip, a bottle of mineral oil, a tube of positive control, a tube of negative control, a tube of diluent and a tube of nuclease-free water, wherein the freeze-drying microchip comprises a primer, a probe, Taq enzyme, reverse transcriptase, trehalose, Tris-Cl, dNTP, Mg2+。
The porcine blue ear classical strain and NADC30-Like strain virus fluorescence freeze-drying microchip PCR detection kit is characterized in that the 3' end of the nucleotide sequence of a Taqman probe is marked with an MGB fluorescence quenching group. The freeze-dried microchip is coated with primers and Taqman probes having the following nucleotide sequences:
the upstream primer of the classical strain of porcine blue ear: 5 '-TGTTGTGACTTGAGCGTCGAT-3',
the downstream primer of the classical strain of porcine blue-ear: 5 '-CAAAAGGCCAGGAACCGTAA-3',
classical strain Taqman probe of porcine blue ear: 5 'FAM-TTTGTGATGCTCGTCAGG-MGB-3'.
NADC30-Like strain upstream primer: 5 '-GCCTCGCTCAGAACTTCCT-3',
NADC 30-downstream primer of Like strain: 5 '-CACCACGATGTAGGCTTCAG-3',
NADC30-Like strain Taqman probe: 5 'ROX-AGCATCTCGTCTCC-MGB-3'.
The final concentration of the freeze-drying system of the fluorescence quantitative PCR comprises: upstream primer 0.4. mu.M; downstream primer 0.4. mu.M; taqman probe 0.4. mu.M; DNA polymerase 0.5U/. mu.L; reverse transcriptase 0.5U/. mu.L; dNTP 0.3 mM; mg (magnesium)2+3 mM; 5 mu M of trehalose; Tris-Cl5 mM; the balance of sterilized deionized water;
preferably, the total volume of the freeze-drying system of the fluorescence quantitative PCR is 36 μ L, and each pore volume is 1.2 μ L;
the number of sample wells on the whole lyophilized microchip of the AriaYSB lyophilized microchip for fluorescent quantitative PCR was 30, and 1.2. mu.L of the above fluorescent PCR reaction system (containing classical porcine blue-ear strain and 2 sets of primer probes of NADC30-Like strain) was added to each microchip well.
The freeze-dried microchip for the fluorescent quantitative PCR was frozen for 1 hour at-80 ℃. The freeze-drying conditions of the equipment are as follows: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the pre-freezing time is kept for 1h, then the equipment is vacuumized, and the freeze drying is kept for 1 h; the drying stage was resolved, the spacer temperature was again raised to-25 ℃ for 1h, then raised to 37 ℃ and held for 2h, and finally lowered to 25 ℃ and held for 1 h.
The porcine blue ear classical strain and NADC30-Like strain virus freeze-drying microchip fluorescent RT-PCR detection kit is characterized in that the freeze-drying microchip fluorescent RT-PCR reaction conditions are as follows: 10min at 50 ℃ as the first step cycle; the second step is circulation at 95 ℃ for 1 min; 95 ℃ for 5s, 60 ℃ for 15s, for a third step, 40 cycles, which was performed at the end of extension for each cycle with detection of the fluorescent signal.
The porcine blue ear classical strain and NADC30-Like strain virus freeze-drying microchip fluorescent RT-PCR detection kit comprises a PCR freeze-drying microchip, wherein the PCR freeze-drying microchip comprises primers and Taqman probes with the following nucleotide sequences: the upstream primer of the classical strain of porcine blue ear: 5 '-TGTTGTGACTTGAGCGTCGAT-3', downstream primer of classical strain of porcine blue ear: 5 '-CAAAAGGCCAGGAACCGTAA-3', classical strain Taqman probe of porcine blue ear: 5 'FAM-TTTGTGATGCTCGTCAGG-MGB-3'; NADC30-Like strain upstream primer: 5 '-GCCTCGCTCAGAACTTCCT-3', and downstream primer of strain NADC 30-Like: 5 '-CACCACGATGTAGGCTTCAG-3', NADC30-Like strain Taqman probe: 5 'ROX-AGCATCTCGTCTCC-MGB-3'.
As a further improvement, the 3' end of the nucleotide sequence of the Taqman probe is labeled with an MGB fluorescence quenching group.
Marking FAM fluorescent reporter group at the 5' end of the nucleotide sequence of the porcine blue-ear classical strain virus detection Taqman probe; the 5' end of the nucleotide sequence of the NADC30-Like strain virus detection Taqman probe is labeled with a ROX fluorescent reporter group.
The lyophilized microchip reagent further comprises DNA polymerase, reverse transcriptase, dNTP, Mg2+Trehalose, Tris-Cl.
The number of wells on the whole lyophilized microchip of the lyophilized AriaYSB microchip for fluorescent quantitative PCR was 30, and the above-mentioned fluorescent PCR reaction system (containing 2 sets of primer probes for classical strain of porcine blue ear and for strain NADC 30-Like) was diluted with 2 Xdiluent, and 1.2. mu.L of each microchip well was added.
The freeze-drying condition is that the freeze-drying microchip of the fluorescent quantitative PCR is frozen for 1h at minus 80 ℃. The freeze-drying conditions of the equipment are as follows: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the pre-freezing time is kept for 1h, then the equipment is vacuumized, and the freeze drying is kept for 1 h; the drying stage was resolved, the spacer temperature was again raised to-25 ℃ for 1h, then raised to 37 ℃ and held for 2h, and finally lowered to 25 ℃ and held for 1 h.
The conventional reagent comprises a commercialized molecular reagent including mineral oil, diluent and nuclease-free water;
the commercial dilution was 10 XBuffer (Mg free)2+) The dilution was 2 Xbuffer using nuclease-free water.
The freeze-drying microchip fluorescence quantitative PCR 1.2 u L reaction system includes: fluorescent quantitative PCR freeze-drying reagent wells and 0.6 muL of diluent; the RNA of the sample was detected at 0.6. mu.L.
The kit also comprises a positive control, wherein the positive control is a virus genome cDNA mixture of a porcine blue-ear classical strain and an NADC30-Like strain.
The porcine blue ear classical strain and NADC30-Like strain virus freeze-drying microchip fluorescent RT-PCR detection kit is characterized in that the primers and/or the kit are used for detecting a sample to be detected.
The detection refers to the fluorescent quantitative PCR detection of the freeze-drying microchip; the upstream primer is 0.4 mu M; downstream primer 0.4. mu.M; taqman probe 0.4. mu.M; DNA polymerase 0.5U/. mu.L; reverse transcriptase 0.5U/. mu.L; dNTP 0.3 mM; mg (magnesium)2+3 mM; 5 mu M of trehalose; Tris-Cl5 mM; the balance was sterile deionized water, with a total volume of 36. mu.L, and a pore volume of 1.2. mu.L.
The reaction conditions of the freeze-drying microchip fluorescence RT-PCR include: 10min at 50 ℃ as the first step cycle; the second step is circulation at 95 ℃ for 1 min; 95 ℃ for 5s, 60 ℃ for 15s, for 40 cycles of the third step, which was performed at the end of extension for each cycle with detection of the fluorescent signal.
Due to the adoption of the technical scheme, the invention at least has the following advantages:
(1) according to the invention, primers are designed according to NSP2 genes of porcine blue-ear classical strains and NADC30-Like strain viruses found in China, specific primers and Taqman-MGB probes are synthesized, the porcine blue-ear classical strains and the NADC30-Like strain viruses can be simultaneously, rapidly and sensitively detected by adopting a fluorescent quantitative PCR method, and the method has high accuracy, specificity and sensitivity and good repeatability.
(2) On one hand, the invention adopts high copy target gene, on the other hand, the invention adopts Taqman-MGB probe fluorescent quantitative PCR detection method, so that the sensitivity of the fluorescent quantitative PCR detection by the Taqman-MGB probe method is about 100 times of that of the common PCR.
(3) As the quantitative detection technology-Taqman-MGB fluorescent quantitative PCR (Real-time PCR) is adopted, the method (Real-time PCR) has the advantages of single-tube closed operation, pollution prevention, high automation degree, strong specificity, Real-time monitoring and the like, and effectively solves the limitation that the traditional method can only carry out end-point detection.
(4) Compared with the currently used fluorescent PCR detection technology, the reaction system of the freeze-drying microchip technology is smaller and is only 1.2 muL, and the conventional reaction system is 20-25 muL, so the microchip technology can save the use amount of RT-PCR amplification reagents and samples.
(5) The smaller reaction system of the freeze-drying microchip can ensure that the system is heated more uniformly and the temperature rise and fall speed is faster in the PCR amplification process. The temperature rise rate (10-12 ℃/S) is higher than that (3-5 ℃/S) of the currently adopted fluorescence PCR detection system. The whole process of the fluorescence PCR (including sample adding) can be completed within 40 minutes, and the computer automatically reports the result without electrophoresis and other subsequent work, thereby facilitating the operation and reducing the pollution.
(6) The freeze-drying microchip can be stored and transported at normal temperature, repeated freezing and thawing of reagents are avoided, and the detection result is more stable.
The Real-time fluorescence quantitative PCR technology (Real-time PCR) adopted by the invention is a method for adding a fluorescent group into a PCR reaction system, monitoring the whole PCR process in Real time by utilizing fluorescent signal accumulation, and finally carrying out quantitative analysis on an unknown template through a standard curve.
The microchip used in the present invention is a reaction area formed of a silica gel or aluminum disk with a microreactor (the volume and mass of the microreactor depend on the type of microchip) covered with a protective film. The reaction zone of the microchip was covered with a layer of mineral oil. The reagent penetrates through a layer of mineral oil and is injected into the microreactor through a manual or automatic pipette with a gun head, so that cross contamination of a detection sample and evaporation of a reaction system can be avoided.
The invention freezes the PCR reaction reagent on the microchip, combines the Taqman probe fluorescent quantitative PCR technology, and the analysis of the nucleic acid is that the microchip reads the fluorescent signal generated by the PCR product on each reactor simultaneously during the thermal cycle. The microchip technology adopted by the invention is different from the current common plastic PCR tube as a carrier to contain a PCR reaction system for PCR amplification, but adopts a metal carrier microchip, and the reaction system and a sample are directly added on the metal carrier for PCR amplification. The heat conduction efficiency and the temperature rise and fall speed of the metal carrier are higher, and the reaction program time can be greatly shortened. The method has the characteristics of small reaction system, high automation degree, short reaction time, strong specificity, high sensitivity, strong repeatability, no need of low-temperature storage of reaction reagents, capability of carrying out quantitative detection, small possibility of pollution caused by microchip operation and the like. The product of the invention can rapidly identify and distinguish porcine blue ear classical strains and NADC30-Like strains under the condition of consistent reaction conditions, has simple operation, and can complete the sample treatment to result analysis within 1 hour.
In conclusion, by adopting the technical scheme, the invention develops a reaction system of microchip fluorescence quantitative PCR capable of quickly and effectively detecting porcine blue ear classical strains and NADC30-Like strains viruses by designing specific primers and probes and optimizing microchip freeze-drying conditions, and simultaneously prepares a detection kit based on the method. A large number of experiments prove that the primer probe/kit/detection method has higher accuracy and excellent specificity compared with the conventional method, and the lowest detection limit can reach 10TCID50The sensitivity is very high when the kit is used for detection, and the intra-batch variation coefficient of the detection kit is between 0.91% and 1.08%, and the inter-batch variation coefficient is between 1.13% and 1.87%, so that the repeatability is good.
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FIG. 1 is a specific assay for the fluorescent RT-PCR assay of classical strain of porcine blue-ear virus on a lyophilized microchip. In the figure, 1: classical strain virus of porcine blue-ear; 2: highly pathogenic classical variant strains; 3: NADC30-Like strain; 4: transmissible gastroenteritis virus of swine; 5: epidemic diarrhea virus; 6: rotavirus; 7: a swine influenza virus; 8: hog cholera virus; 9: a pig torus; 10: the pigs are small; 11: porcine pseudorabies nucleic acid; 12: nuclease-free water.
FIG. 2 is the specific detection of porcine blue ear NADC30-Like strain virus by the fluorescent RT-PCR detection of the freeze-drying microchip. In the figure, 1: NADC30-Like strain; 2: highly pathogenic classical variant strains; 3: classical strain virus of porcine blue-ear; 4: transmissible gastroenteritis virus of swine; 5: epidemic diarrhea virus; 6: rotavirus; 7: a swine influenza virus; 8: hog cholera virus; 9: a pig torus; 10: the pigs are small; 11: porcine pseudorabies nucleic acid; 12: nuclease-free water.
FIG. 3 shows the result of detecting the virus sensitivity of classical porcine blue-ear strains by using the freeze-dried microchip fluorescent RT-PCR reagent. In the figure, A: 1X 106TCID50/mL；B：1×105TCID50/mL；C：1×104TCID50/mL；D：1×103TCID50/mL；E：1×102TCID50/mL；F：1×101TCID50/mL；G:1×100TCID50Per mL; h, nuclease-free water.
FIG. 4 shows the result of detecting the virus sensitivity of classical porcine blue-ear strains by using a conventional fluorescent RT-PCR reagent. In the figure, A: 1X 106TCID50/mL；B：1×105TCID50/mL；C：1×104TCID50/mL；D：1×103TCID50/mL；E：1×102TCID50/mL；F：1×101TCID50/mL；G:1×100TCID50Per mL; h, nuclease-free water.
FIG. 5 shows the standard curve of the porcine blue-ear classical strain virus detected by the freeze-dried microchip fluorescent RT-PCR reagent.
FIG. 6 shows the standard curve result of conventional fluorescent RT-PCR reagent for detecting classical strain virus of porcine blue-ear.
Detailed Description
In order to make the technical means of the present invention more clearly understood, the present invention is further described in detail with reference to the accompanying drawings and the detailed description. Unless otherwise indicated, the reagents used in the following examples are analytical grade reagents and are commercially available from a regular channel.
Sources or documentations of biological materials
Classical strains (VR-2322 strain) and an NADC30-Like strain used in experimental examples of the porcine reproductive and respiratory syndrome virus are standard inactivated strains for national animal epidemic control and can be obtained commercially. Other strains are also commercially available.
The group of embodiments provides a freeze-drying microchip for identifying classical strains of porcine reproductive and respiratory syndrome virus and an NADC30-Like strain, which is characterized in that a fluorescence PCR reaction system for identifying classical strains of porcine reproductive and respiratory syndrome and an NADC30-Like strain is fixed on the microchip by freeze-drying;
the fluorescent PCR reaction system comprises: the following primers and probes:
classical strain upstream primer of porcine reproductive and respiratory syndrome virus: 5 '-TGTTGTGACTTGAGCGTCGAT-3',
classical strain downstream primers for porcine reproductive and respiratory syndrome virus: 5 '-CAAAAGGCCAGGAACCGTAA-3',
classical strain Taqman probes for porcine reproductive and respiratory syndrome virus: 5 '-TTTGTGATGCTCGTCAGG-3';
NADC30-Like strain upstream primer: 5 '-GCCTCGCTCAGAACTTCCT-3',
NADC 30-downstream primer of Like strain: 5 '-CACCACGATGTAGGCTTCAG-3',
NADC30-Like strain Taqman probe: 5 '-AGCATCTCGTCTCC-3'.
In a specific embodiment, the fluorescent PCR reaction system further comprises: taq enzyme, reverse transcriptase, trehalose, Tris-Cl, dNTP, Mg2+；
Preferably, the 5' end of the Taqman probe of classical strain and NADC30-Like strain of the porcine reproductive and respiratory syndrome virus is marked with a fluorescent reporter group; the 3' end is marked with a fluorescence quenching group.
In a more specific embodiment, the fluorescent PCR reaction system comprises: upstream primer 0.4. mu.M; downstream primer 0.4. mu.M; taqman probe 0.4. mu.M; DNA polymerase 0.5U/. mu.L; reverse transcriptase 0.5U/. mu.L; dNTP 0.3 mM; mg (magnesium)2+3 mM; 5 mu M of trehalose; Tris-Cl5mM, and the balance of sterilized deionized water;
the final concentrations of these components were either those of the fluorescent PCR reaction system before lyophilization or those of the fluorescent PCR reaction system after lyophilization after addition of 0.6uL of 2 Xdilution of the sample.
The total volume of the fluorescent PCR reaction system prepared before lyophilization was typically 36. mu.L, with a volume of 1.2. mu.L per well.
Preferably, the fluorescence reporter group marked at the 5' end of the Taqman probe of classical strain of porcine reproductive and respiratory syndrome virus is a FAM fluorescence reporter group;
the fluorescence reporter group marked at the 5' end of the Taqman probe of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) NADC30-Like strain is a ROX fluorescence reporter group;
the fluorescence quenching group marked at the 3' end of the Taqman probe of classical strain and NADC30-Like strain of the porcine reproductive and respiratory syndrome virus is MGB quenching group.
In some embodiments, a plurality of wells are disposed on the microchip; the fluorescent PCR reaction system is fixed in the sample adding hole by freeze-drying;
preferably, the microchip on the sample hole is 30; the structure of the microchip is matched with the structure of a sample adding plate of the PCR instrument. The structure of the microchip is similar to that of a PCR plate, but the microchip of the invention is uncovered, mineral oil is needed for sealing the sample adding hole, and the hole is not easy to be mixed and polluted during sample adding. Therefore, the phenomenon of flow overflow or cross-hole contamination of the PCR system can not occur.
In other embodiments, the lyophilization comprises the steps of: freezing the microchip filled with the fluorescent PCR reaction system at-80 ℃ for 1h, and then carrying out equipment freeze-drying;
the microchip containing the fluorescent PCR reaction system is frozen at-80 ℃ for 1h in advance for pre-freezing, so that the system can be kept in a solid state in the first pre-freezing stage, and the drying time can be shortened in the step.
The equipment freeze-drying refers to that freeze-drying is carried out in a special freeze-drying equipment by setting the working conditions of the equipment; preferably, the freeze-drying apparatus is a vacuum freeze-dryer.
Preferably, the device lyophilization comprises: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the pre-freezing time is kept for 1h, then the equipment is vacuumized, and the freeze drying is kept for 1 h; the drying stage was resolved, the spacer temperature was again raised to-25 ℃ for 1h, then raised to 37 ℃ and held for 2h, and finally lowered to 25 ℃ and held for 1 h.
The temperature of the partition plate is the temperature of a tray partition plate in a freeze dryer, and a microchip filled with the fluorescent PCR reaction system is required to be placed on the tray partition plate; the pre-freezing stage is used to maintain the fluorescent PCR reaction system in a solid state. The main purpose of the step of vacuumizing equipment and keeping freeze drying for 1h is to vacuum a freeze dryer, and the internal air needs to be pumped to dry so as to sublimate the water in the solid, thereby achieving the freeze drying effect. The following 2 steps of increasing the temperature of the clapboard to-25 ℃ for 1h and increasing the temperature of the clapboard to 37 ℃ for 2h are both keeping the drying and sublimation processes; finally, the function of "lowering the partition to 25 ℃ and keeping it for 1 h" is to keep the freeze-dried product stable at 25 ℃, so that the whole freeze-drying process is basically completed.
The above-mentioned "equipment freeze-drying" process is a set of freeze-drying process originally invented by said invention for microchip equipped with said fluorescent PCR reaction system. Due to different eutectic points of the freeze-drying reagents, the eutectic points of the freeze-drying reagents need to be measured respectively, and the temperature rise time in the drying process and the drying time in the drying process need to be optimized. The effect of the components within the lyophilized reagent on the eutectic point was explored by adjusting the reagent component concentrations. Particularly, the enzyme in the reaction system is stored for a long time at normal temperature, the activity of the enzyme is reduced, and a protective agent and a stabilizing agent component, such as trehalose, are required to be added for 5 mu M; the Tris-Cl5mM, and whether the concentration thereof inhibits the PCR reaction or not can be verified and adjusted by experiments, so that the excellent effects of high sensitivity, high repeatability, high accuracy, high specificity and the like recorded by the invention can be obtained when the freeze-drying microchip is used for carrying out fluorescence PCR detection. In addition, the reconstitution effect of the diluent used after the freeze-drying process is carried out is also required to be considered, and the freeze-drying process is also required to be adjusted so that the freeze-drying microchip can achieve the final excellent effects of high sensitivity, high repeatability, high accuracy, high specificity and the like obtained by the detection method.
The skilled person can synthesize the primers artificially according to the disclosure of the present invention, and use them for qualitative or quantitative detection of classical porcine reproductive and respiratory syndrome virus and NADC30-Like strain virus, to obtain the expected effect according to the present invention, therefore any primers synthesized for commercial purposes and placed in the commercial package box labeled for "detecting classical porcine reproductive and respiratory syndrome virus and NADC30-Like strain virus" or primers using the above sequences for commercial detection of classical porcine reproductive and respiratory syndrome virus and NADC30-Like strain virus fall within the scope of the present invention.
This group of embodiments provides a kit for identifying classical strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) from strains of NADC30-Like, comprising the lyophilization microchip of any one of group 1 embodiments.
In a further embodiment, the kit further comprises: and the diluent is used for dripping into a sample adding hole of the freeze-drying microchip, and then the freeze-drying microchip is placed on a fluorescent PCR instrument for fluorescent PCR amplification.
The diluent is PCR 10 × buffer, and the specific components do not contain Mg2+This dilution is commercially available in the presence of 500mM KCl, 100mM Tris-HCl, 0.1% gelatin. When used, the diluted solution was diluted to 2 Xbuffer using nuclease-free water, and 0.6. mu.L of the diluted solution was added to each well of the microchip.
In still further embodiments, the kit further comprises: and the mineral oil is used for sealing the sample adding hole on the microchip, and the sample is not easily polluted because the system is very micro and the oil is not sealed, which is equivalent to a tube cover for covering the PCR tube and is easily dried by air.
Positive control, specifically, a genomic cDNA mixture of classical porcine reproductive and respiratory syndrome virus strains and an NADC30-Like strain;
negative control, specifically nuclease-free water.
In specific embodiments, the probe is labeled at the 3 'end with an MGB quencher and at the 5' end with a FAM or ROX fluorescent reporter. The above-mentioned "MGB", "FAM" and "ROX" groups are all fluorescent groups commonly used in the art, and those skilled in the art can also select other fluorescence quenching groups and fluorescence reporter groups commonly used in the art to replace the "MGB", "FAM" and "ROX" groups herein, for example, common fluorescence quenching groups also include: BHQ-1, BHQ-2, Dabcyl 2; common fluorescent reporter groups may also be selected from: TET, HEX, 5-TAMRA, Texas Red-X, Cy3 (TYTM 563), Cy5 (TYTM 665), JOE.
In a further embodiment, the kit further comprises an AriaYSB microchip and conventional reagents for performing fluorescent quantitative PCR assays.
The lyophilized microchip reagent further comprises DNA polymerase, reverse transcriptase, dNTP, Mg2+Trehalose, Tris-Cl.
In a preferred embodiment, the conventional reagents include DNA polymerase, reverse transcriptase, dNTP, Mg2+Trehalose, Tris-Cl; and/or, a commercial molecular reagent comprising said mineral oil, diluent, nuclease-free water;
in a further preferred embodiment, the commercial dilution is 10 × buffer (Mg-free)2+) The dilution was 2 Xbuffer using nuclease-free water. There are many more commercially available DNA polymerases, reverse transcriptases, dNTPs, and 10 XBuffers in the field, and those skilled in the art can select other brands and other types of molecular reagents.
The group of embodiments provides a freeze-drying microchip fluorescence RT-PCR detection method for identifying porcine blue ear classical strains and NADC30-Like strains. All the examples of this group have the following characteristics: the method comprises the following steps: performing fluorescence PCR amplification on a sample to be tested by using the freeze-dried microchip described in any one of the group 1 embodiments and/or the kit described in any one of the group 2 embodiments.
In some embodiments, after the sample to be tested and the diluent are added into the sample adding hole of the freeze-drying microchip, the freeze-drying microchip is placed on a fluorescent PCR instrument for the fluorescent PCR amplification;
preferably, the addition amount of the diluent 2 x buffer is 0.6 μ L, and the addition amount of the positive control, the negative control or the sample is 0.6 μ L;
further preferably, the reaction procedure of the fluorescent PCR amplification is: 10min at 50 ℃; 1min at 95 ℃; the fluorescence signal was detected at the end of extension in 40 cycles of 1 cycle of 95 ℃ 5s and 60 ℃ 15 s.
In some embodiments, the detection is a fluorescent quantitative PCR detection; the freeze-drying microchip of the classical porcine blue ear strain and the strain NADC30-Like comprises: upstream primer 0.4. mu.M; downstream primer 0.4. mu.M; taqman probe 0.4. mu.M; DNA polymerase 0.5U/. mu.L; reverse transcriptase 0.5U/. mu.L; dNTP 0.3 mM; mg (magnesium)2+3 mM; 5 mu M of trehalose; Tris-Cl5 mM; the balance was sterile deionized water, with a total volume of 36. mu.L, and a pore volume of 1.2. mu.L.
The number of sample wells on the whole freeze-dried microchip of the fluorescence quantitative PCR is 30, and 1.2 mu L of the fluorescence quantitative PCR system (containing 2 sets of primer probes of classical porcine blue ear strain and NADC30-Like strain) is added into each well.
In some embodiments, the lyophilization conditions are that the lyophilized microchip for fluorescent quantitative PCR is frozen for 1h prior to-80 ℃. The freeze-drying conditions of the equipment are as follows: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the pre-freezing time is kept for 1h, then the equipment is vacuumized, and the freeze drying is kept for 1 h; the drying stage was resolved, the spacer temperature was again raised to-25 ℃ for 1h, then raised to 37 ℃ and held for 2h, and finally lowered to 25 ℃ and held for 1 h.
In a further embodiment, the reaction procedure of the fluorescent quantitative PCR comprises: 10min at 50 ℃ as the first step cycle; the second step is circulation at 95 ℃ for 1 min; 95 ℃ for 5s, 60 ℃ for 15s, for 40 cycles of the third step, which was performed at the end of extension for each cycle with detection of the fluorescent signal.
Examples of the experiments1 porcine blue ear classical strain and NADC30-Like strain virus freeze-drying microchip fluorescent RT-PCR detection kit
Specificity verification
1. Design of primers and Taqman-MGB probes
According to the domestic detection of the porcine reproductive and respiratory syndrome classical strain and the NADC30-Like strain virus, the specific conserved sequences of the porcine reproductive and respiratory syndrome classical strain and the NADC30-Like strain virus NSP2 gene are found, and a plurality of pairs of primers and probes are designed. Finally, respectively determining a group of optimal primers and a Taqman-MGB probe through comparison and screening.
The upstream primer of the classical strain of porcine blue ear: 5' -TGTTGTGACTTGAGCGTCGAT-3
The downstream primer of the classical strain of porcine blue-ear: 5' -CAAAAGGCCAGGAACCGTAA-3
Classical strain Taqman probe of porcine blue ear: 5 'FAM-TTTGTGATGCTCGTCAGG-MGB-3';
NADC30-Like strain upstream primer: 5' -GCCTCGCTCAGAACTTCCT-3
NADC 30-downstream primer of Like strain: 5' -CACCACGATGTAGGCTTCAG-3
NADC30-Like strain Taqman probe: 5 'ROX-AGCATCTCGTCTCC-MGB-3'.
Wherein, FAM fluorescent reporter group is marked at the 5' end of the nucleotide sequence of the classical strain Taqman probe of the porcine reproductive and respiratory syndrome virus; the 5' end of the nucleotide sequence of the NADC30-Like strain Taqman probe is marked with ROX fluorescent reporter group. MGB quenching fluorescent group is marked at the 3' end. The reason why MGB is selected by quenching the fluorescent group is that the TaqMan-MGB probe has the following advantages compared with the conventional TaqMan-TAMRA probe: (1) increasing the TM value, on average 15bases can increase 18 ℃, which can shorten the length of the probe, greatly aid in designing sequences with high AT content, and increase the TM value difference between paired and unpaired templates. (2) And the signal to noise ratio is improved, because the quenching group at the 3' end of the probe is a non-luminous fluorescent group and is closer to the position of the reporter group in space, the experimental result is more accurate, and the resolution ratio is higher.
2. Viral RNA extraction
The total RNA extraction kit (Beijing Yishengbao biotechnology limited, product number EAR002) is used for extracting the genome RNA of highly pathogenic classical variant strains, NADC30-Like strains, classical strain viruses of porcine reproductive and respiratory syndrome, transmissible gastroenteritis viruses of swine, epidemic diarrhea viruses, rotavirus, swine influenza viruses and classical swine fever viruses. The total DNA extraction kit (product number EAD001, Beijing Yishengbao Biotechnology Co., Ltd.) was used to extract the genome DNA of porcine circulus, porcine parvovirus and porcine pseudorabies. Placing at-20 ℃ for later use.
3. Freeze-drying microchip preparation
The lyophilized microchip RT-PCR system was prepared according to the following reaction system:
the entire lyophilized microchip of the fluorescence quantitative PCR AriaYSB microchip (Beijing Yishengbao Biotech Co., Ltd.) was spotted with 30 wells, and 1.2. mu.L of the above fluorescence quantitative PCR system (containing 2 sets of primer probes for classical strain of porcine Lancet and for strain NADC 30-Like) was added to each well.
The microchip coated with the PCR reagents was frozen for 1 hour at-80 ℃. The freeze-drying conditions of the equipment are as follows: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the pre-freezing time is kept for 1h, then the equipment is vacuumized, and the freeze drying is kept for 1 h; the drying stage was resolved, the spacer temperature was again raised to-25 ℃ for 1h, then raised to 37 ℃ and held for 2h, and finally lowered to 25 ℃ and held for 1 h.
4. Freeze-drying microchip specificity verification
Add 10 x buffer dilution to 24 u L nuclease free water after shaking mixing, each hole add 0.6u L, on the microchip surface add 600 u L mineral oil, until it covers all the loading hole. Then 0.6 mu L of highly pathogenic classical variant strain, an NADC30-Like strain, porcine blue ear classical strain virus, porcine transmissible gastroenteritis virus, epidemic diarrhea virus, rotavirus, swine influenza virus, swine fever virus, porcine circus, porcine parvovirus and porcine pseudorabies nucleic acid extract are respectively added, and 0.6 mu L of nuclease-free water is added into the last hole to be used as a negative control.
The conditions for the lyophilized microchip fluorescence RT-PCR reaction were as follows: 10min at 50 ℃ as the first step cycle; the second step is circulation at 95 ℃ for 1 min; 95 ℃ for 5s, 60 ℃ for 15s, for 40 cycles of the third step, which was performed at the end of extension for each cycle with detection of the fluorescent signal.
The fluorescent reporter groups marked by the probe are 2, namely a FAM fluorescent reporter group and a ROX fluorescent reporter group, and corresponding fluorescent reporter groups can be respectively selected to display results when the results are analyzed by using fluorescent PCR software, for example, the FAM fluorescent reporter group probe, namely the probe of classical strain of porcine reproductive and respiratory syndrome virus, only displays the detection results of the primer probe of the classical strain of porcine reproductive and respiratory syndrome virus. The ROX probe (channel) is a probe of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) NADC30-Like strain, and only shows the detection result of the primer probe of the PRRSV NADC30-Like strain. The results for the 2 fluorescent reporter groups can be shown separately.
The experimental result is shown in the figure, the freeze-drying microchip is coated with double fluorescent RT-PCR reagents of the porcine reproductive and respiratory syndrome Virus classical strain and the NADC30-Like strain, the result in the figure 1 shows that the porcine reproductive and respiratory syndrome Virus RNA detected by the porcine reproductive and respiratory syndrome classical strain virus channel is positive amplification, the Ct value is 22.93, and an amplification curve is provided. Highly pathogenic classical variants, strain NADC30-Like, transmissible gastroenteritis virus, epidemic diarrhea virus, rotavirus, swine influenza virus, classical swine fever virus, porcine circovirus, porcine parvovirus, porcine pseudorabies and negative control samples were not amplified non-specifically. FIG. 2 shows that the NADC30-Like strain channel detects the positive amplification of the NADC30-Like strain RNA, the Ct value is 24.09, and an amplification curve exists. The highly pathogenic classical variant strain, porcine blue ear classical strain virus, porcine transmissible gastroenteritis virus, epidemic diarrhea virus, rotavirus, swine influenza virus, swine fever virus, porcine circulus, porcine parvovirus, porcine pseudorabies and negative control sample have no non-specific amplification. The results obtained were in full agreement with expectations. It can be seen from the amplification curves of all samples that the curves overlap well in the early stage of amplification, especially around the fluorescence threshold.
Experimental example 2 porcine Lanchow classical strain and NADC30-Like strain virus freeze-drying microchip fluorescent RT-PCR detection kit
Sensitivity verification and comparison with conventional fluorescent RT-PCR reagent
The concentration is 1 x 107TCID50Per mL of classical strain of porcine blue-ear virus solution, and gradient of 10 timesAnd (6) diluting. Extraction of 1X 106TCID50/mL～1×100TCID50And taking the genomic RNA of each gradient concentration porcine reproductive and respiratory syndrome classical strain virus as a template and nuclease-free water as negative control, and performing freeze-drying microchip fluorescent RT-PCR reagent and conventional fluorescent RT-PCR reagent to detect the universal sensitivity detection of the porcine reproductive and respiratory syndrome classical strain virus.
The lyophilized microchip fluorescent RT-PCR system was prepared as in example 1 by adding 10 XBuffer diluent to 24. mu.L of nuclease-free water, shaking and mixing, adding 0.6. mu.L of the diluent to each well, adding 600. mu.L of mineral oil to the surface of the microchip, and allowing the microchip to cover all the wells. Adding 1 × 10 of the mixture into the holes respectively6TCID50/mL～1×100TCID50The genomic RNA of porcine reproductive and respiratory syndrome virus was added to 1 well of each of the gradient concentrations, and 0.6. mu.L of nuclease-free water was added to the final well to serve as a negative control.
The conditions for the lyophilized microchip fluorescence RT-PCR reaction were as follows: 10min at 50 ℃ as the first step cycle; the second step is circulation at 95 ℃ for 1 min; 95 ℃ for 5s, 60 ℃ for 15s, for 40 cycles of the third step, which was performed at the end of extension for each cycle with detection of the fluorescent signal.
Preparing a conventional fluorescent RT-PCR reagent system:
conventional fluorescent RT-PCR is as follows: 30min at 50 ℃, which is the first circulation; 5min at 95 ℃ as a second step cycle; 95 ℃ for 15s, 60 ℃ for 45s, for 40 cycles of the third step, which was performed at the end of extension for each cycle with detection of the fluorescent signal.
FIG. 3 shows the results of the freeze-dried microchip fluorescence RT-PCR with the lowest assay sample concentration of 1X 101TCID50The Ct value is 33.97 for mL, so the reaction cycle number of 40 can greatly meet the minimum detection requirement. As can be seen from the amplification curves of the starting templates with different concentrations, the baseline of the curve is flat, the exponential region is obvious, and the slope is large, which all indicate that the amplification of the template is more ideal under the conditions. FIG. 4 shows the results of conventional fluorescence RT-PCR with the lowest detected sample concentration of 1X 102TCID50/mL，Ct value is 34.57. The results show that the sensitivity of the fluorescence RT-PCR detection of the freeze-drying microchip is higher (about 10 times higher) than that of the conventional fluorescence RT-PCR detection. Because the Ct value has a linear relation with the logarithm of the initial template, a standard curve can be made by using the Ct value and the real concentration of the template, and a linear relation equation is listed. The results in FIG. 5 and FIG. 6 show that the freeze-dried microchip fluorescence RT-PCR amplification efficiency is 105.40%, the conventional fluorescence RT-PCR amplification efficiency is 87.58%, and the freeze-dried microchip fluorescence RT-PCR amplification efficiency is higher. The standard curve formula and the correlation coefficient of the two are relatively close. The correlation coefficient of the freeze-drying microchip fluorescence RT-PCR and the conventional fluorescence RT-PCR is 0.99, so that the correlation of the freeze-drying microchip fluorescence RT-PCR and the conventional fluorescence RT-PCR is good. The fluorescent RT-PCR reaction system of the freeze-drying microchip is 1.2 mu L, the conventional fluorescent RT-PCR system is 25 mu L, and the fluorescent RT-PCR reaction system of the freeze-drying microchip is reduced by more than 20 times. The freeze-dried microchip fluorescence RT-PCR reaction time is less than 40 minutes, the conventional fluorescence RT-PCR reaction time is 120 minutes, and the running time is shortened by 2/3.
Experimental example 3: porcine blue ear classical strain and NADC30-Like strain virus freeze-drying microchip fluorescent RT-PCR detection kit
Preparation and detection of
1. Preparation of the kit:
preparation of lyophilized microchip:
the lyophilized microchip RT-PCR system was prepared according to the following reaction system:
the total number of wells on the lyophilized microchip of the AriaYSB microchip for fluorescent quantitative PCR (Beijing Yishengbao Biotech Co., Ltd.) was 30, and 1.2. mu.L of each well of the above fluorescent quantitative PCR system (containing 2 sets of primer probes for classical strain of porcine Lancete and strain NADC 30-Like) was added.
The microchip coated with the PCR reagents was frozen for 1 hour at-80 ℃. The freeze-drying conditions of the equipment are as follows: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the pre-freezing time is kept for 1h, then the equipment is vacuumized, and the freeze drying is kept for 1 h; the drying stage was resolved, the spacer temperature was again raised to-25 ℃ for 1h, then raised to 37 ℃ and held for 2h, and finally lowered to 25 ℃ and held for 1 h.
Reagent 1: diluent Taq Buffer (10 ×) (Thermo Scientific, cat # B650060), 6 μ L;
reagent 2: 1mL of mineral oil (Sangon Biotech, cat # A630217);
reagent 3: positive control (NADC30-Like strain, classical strain of porcine blue-ear virus genome cDNA mixture) 30. mu.L;
reagent 4: negative control (no nuclease) 30 μ L;
and (5) reagent: nuclease-free water 50. mu.L.
2. Reproducibility analysis of the kit
3 samples with known positive are selected and subjected to batch repeated detection and batch repeated detection respectively. In-batch duplicate detection: 3 known positive samples were run in the same batch of experiments, with 3 replicates per sample set up. Batch-to-batch repeat experiments: 3 known positive samples were tested in batches, each sample tested individually, with 3 replicates per sample set up.
The fluorescent RT-PCR reaction system of each freeze-drying microchip is 1.2 mu L: before reagent 1 is used, 24. mu.L of reagent 5 is sucked and added into reagent 1, and after shaking and mixing, 0.6. mu.L of reagent is added into each well. Add 600. mu.L of reagent 2 to the microchip surface until it covers all wells. Each well was then filled with 0.6. mu.L of each of the positive sample, reagent 3 (positive control) or reagent 4 (negative control).
The conditions for the lyophilized microchip fluorescence RT-PCR reaction were as follows: 10min at 50 ℃ as the first step cycle; the second step is circulation at 95 ℃ for 1 min; and (3) the temperature is 95 ℃ for 5s, the temperature is 60 ℃ for 15s, the temperature is 40 cycles, the fluorescence signal detection is carried out at the end of the extension of each cycle of the third step, and the experimental result is recorded.
As can be seen from the detection results in Table 1, the intra-batch variation coefficient is between 0.91% and 1.08%, and the inter-batch variation coefficient is between 1.13% and 1.87%, which indicates that the kit has good repeatability.
TABLE 1 kit repeatability analysis
By adopting the technical scheme, the invention develops a reaction system for microchip fluorescent quantitative PCR detection capable of quickly and effectively detecting porcine blue ear classical strains and NADC30-Like strain viruses by designing specific primers and probes and optimizing microchip freeze-drying conditions, and simultaneously prepares a detection kit based on the method.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention in any way, and it will be apparent to those skilled in the art that the above description of the present invention can be applied to various modifications, equivalent variations or modifications without departing from the spirit and scope of the present invention.
SEQUENCE LISTING
<110> Beijing Yishengbao Biotechnology Ltd
Chinese animal epidemic prevention control center
<120> lyophilization microchip, kit and method for identifying classical strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and strain NADC30-Like
<130>P180494/YSB
<160>6
<170>PatentIn version 3.5
<210>1
<211>21
<212>DNA
<213>Artificial Sequence
<220>
<223> porcine reproductive and respiratory syndrome virus classical strain upstream primer
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<212>DNA
<213>Artificial Sequence
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<223> porcine reproductive and respiratory syndrome virus classical strain downstream primer
<400>2
<210>3
<211>18
<212>DNA
<213>Artificial Sequence
<220>
<223> classical strain Taqman probe sequence of porcine reproductive and respiratory syndrome virus
<400>3
tttgtgatgc tcgtcagg 18
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<211>19
<212>DNA
<213>Artificial Sequence
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<223> upstream primer of NADC30-Like strain
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<212>DNA
<213>Artificial Sequence
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<223> downstream primer of NADC30-Like strain
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<213>Artificial Sequence
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<223> NADC30-Like strain Taqman probe sequence
<400>6
Claims (10)
1. A freeze-drying microchip for identifying classical strains of porcine reproductive and respiratory syndrome virus and an NADC30-Like strain, which is characterized in that a fluorescent PCR reaction system for identifying classical strains of porcine reproductive and respiratory syndrome virus and an NADC30-Like strain is fixed on the microchip by freeze-drying;
the fluorescent PCR reaction system comprises: the following primers and probes:
classical strain upstream primer of porcine reproductive and respiratory syndrome virus: 5 '-TGTTGTGACTTGAGCGTCGAT-3',
classical strain downstream primers for porcine reproductive and respiratory syndrome virus: 5 '-CAAAAGGCCAGGAACCGTAA-3',
classical strain Taqman probe sequence for porcine reproductive and respiratory syndrome virus: 5 '-TTTGTGATGCTCGTCAGG-3';
NADC30-Like strain upstream primer: 5 '-GCCTCGCTCAGAACTTCCT-3',
NADC 30-downstream primer of Like strain: 5 '-CACCACGATGTAGGCTTCAG-3',
NADC30-Like strain Taqman probe sequence: 5 '-AGCATCTCGTCTCC-3'.
2. The lyophilization microchip of claim 1, wherein the fluorescent PCR reaction system further comprises: taq enzyme, reverse transcriptase, trehalose, Tris-Cl, dNTP, Mg2+；
Preferably, the 5' end of the Taqman probe of classical strain and NADC30-Like strain of the porcine reproductive and respiratory syndrome virus is marked with a fluorescent reporter group; the 3' end is marked with a fluorescence quenching group.
3. The lyophilization microchip according to claim 1 or 2, wherein the fluorescent PCR reaction system comprises: upstream primer 0.4. mu.M; downstream primer 0.4. mu.M; taqman probe 0.4. mu.M; DNA polymerase 0.5U/. mu.L; reverse transcriptase 0.5U/. mu.L; dNTP 0.3 mM; mg (magnesium)2+3 mM; 5 mu M of trehalose; Tris-Cl5mM and the balance of sterilized deionized water, wherein the total volume is 36 mu L, and the pore volume is 1.2 mu L;
preferably, the fluorescence reporter group marked at the 5' end of the Taqman probe of classical strain of porcine reproductive and respiratory syndrome virus is a FAM fluorescence reporter group;
the fluorescence reporter group marked at the 5' end of the Taqman probe of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) NADC30-Like strain is a ROX fluorescence reporter group;
the fluorescence quenching group marked at the 3' end of the Taqman probe of classical strain and NADC30-Like strain of the porcine reproductive and respiratory syndrome virus is MGB quenching group.
4. The lyophilization microchip of any one of claims 1 to 3, wherein a plurality of wells are provided on the microchip; the fluorescent PCR reaction system is fixed in the sample adding hole by freeze-drying;
preferably, the microchip on the sample hole is 30; the structure of the microchip is matched with the structure of a sample adding plate of the PCR instrument.
5. The lyophilization microchip of any one of claims 1 to 4, wherein the lyophilization comprises the steps of: freezing the microchip filled with the fluorescent PCR reaction system at-80 ℃ for 1h, and then carrying out equipment freeze-drying;
preferably, the device lyophilization comprises: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the pre-freezing time is kept for 1h, then the equipment is vacuumized, and the freeze drying is kept for 1 h; the drying stage was resolved, the spacer temperature was again raised to-25 ℃ for 1h, then raised to 37 ℃ and held for 2h, and finally lowered to 25 ℃ and held for 1 h.
6. Kit for the identification of classical strains of porcine reproductive and respiratory syndrome virus from strain NADC30-Like, comprising a freeze-dried microchip according to any one of claims 1 to 5.
7. The kit of claim 6, further comprising: the diluent, 10 Xdiluent, was diluted to 2 Xdiluent with nuclease-free water for dripping into the wells of the freeze-dried microchip, and the freeze-dried microchip was subjected to fluorescent PCR amplification on a fluorescent PCR instrument.
8. The kit of claim 6 or 7, further comprising: mineral oil, close the freeze-drying microchip on the sample hole;
positive control, specifically, a genomic cDNA mixture of classical porcine reproductive and respiratory syndrome virus strains and an NADC30-Like strain;
negative control, specifically nuclease-free water.
9. Method for the identification of classical strains of porcine reproductive and respiratory syndrome virus from strain NADC30-Like, characterized in that a sample to be tested is subjected to fluorescent PCR amplification using a freeze-dried microchip according to any one of claims 1 to 5 and/or a kit according to any one of claims 6 to 8.
10. The method according to claim 9, wherein after the sample to be tested and the diluent are added to the sample adding hole of the freeze-dried microchip, the freeze-dried microchip is placed on a fluorescent PCR instrument for the fluorescent PCR amplification;
preferably, the diluent is 10 × buffer;
further preferably, the reaction procedure of the fluorescent PCR amplification is: 10min at 50 ℃; 1min at 95 ℃; the fluorescence signal was detected at the end of extension in 40 cycles of 1 cycle of 95 ℃ 5s and 60 ℃ 15 s.
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|---|---|---|---|---|
| CN112725528A (en) * | 2020-12-31 | 2021-04-30 | 华南农业大学 | Primer group, probe and kit for detecting Porcine Reproductive and Respiratory Syndrome (PRRS) NADC30-like strain |
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| CN114717361A (en) * | 2022-04-08 | 2022-07-08 | 漳州傲农现代农业开发有限公司 | Fluorescent probe primer and kit for porcine reproductive and respiratory syndrome (NADP) NADC30 strain and application of fluorescent probe primer and kit |
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