CN110885900B - Freeze-dried microchip, kit and method for identifying classical strain of porcine reproductive and respiratory syndrome virus and NADC30-Like strain - Google Patents
Freeze-dried microchip, kit and method for identifying classical strain of porcine reproductive and respiratory syndrome virus and NADC30-Like strain Download PDFInfo
- Publication number
- CN110885900B CN110885900B CN201811052502.3A CN201811052502A CN110885900B CN 110885900 B CN110885900 B CN 110885900B CN 201811052502 A CN201811052502 A CN 201811052502A CN 110885900 B CN110885900 B CN 110885900B
- Authority
- CN
- China
- Prior art keywords
- strain
- microchip
- nadc30
- respiratory syndrome
- porcine reproductive
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a freeze-dried microchip, a kit and a method for identifying classical strains of porcine reproductive and respiratory syndrome virus and NADC30-Like strains, belonging to the field of molecular detection. The freeze-dried microchip is characterized in that: a fluorescent PCR reaction system for identifying classical strains of porcine reproductive and respiratory syndrome virus and NADC30-Like strains is immobilized 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 porcine reproductive and respiratory syndrome virus, a downstream primer of a classical strain of porcine reproductive and respiratory syndrome virus, and a Taqman probe sequence of the classical strain of porcine reproductive and respiratory syndrome virus; NADC30-Like strain upstream primer, NADC30-Like strain downstream primer and NADC30-Like strain Taqman probe. The detection kit can detect porcine reproductive and respiratory syndrome virus and NADC30-Like strain simultaneously, and has the advantages of high detection accuracy, high specificity and high sensitivity and short detection time.
Description
Technical Field
The invention relates to the technical field of molecular biological detection of viruses, in particular to a freeze-dried microchip, a kit and a method for identifying classical strains of porcine reproductive and respiratory syndrome virus and NADC30-Like strains.
Background
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is commonly known as "porcine reproductive and respiratory syndrome". The disease is an important epidemic disease of pig reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) which causes reproductive and respiratory disturbance. PRRSV affects mainly pigs, especially sows, and the disease seriously affects their reproductive function, and is characterized clinically by abortion, stillbirth, mummy, weak fetus, dyspnea, and transient cyanosis of the skin of both ears during the onset of disease, so also called blue ear disease. The disease is the first disease which damages the pig farm in recent years, not only can cause abortion, stillbirth, abnormal oestrus and immunosuppression of the sow, but also can cause diarrhea and respiratory diseases of commercial pigs, and seriously affects the growth speed and health of the pigs. The porcine reproductive and respiratory syndrome virus strain has certain pathogenicity, but lower toxicity, and is the best choice for preventing and treating highly pathogenic porcine reproductive and respiratory syndrome virus as a porcine reproductive and respiratory syndrome virus live vaccine. NADC30 strain is first found in China in 2013, then is subjected to continuous mutation, is recombined with domestic porcine reproductive and respiratory syndrome virus strain, is named as NADC30-Like strain, and has stronger toxicity of a few NADC30-Like strains. In the country, NADC was from 2015, a scholars conducted epidemiological investigation of outbreaks of the NADC30-Like strain of blue-ear disease. Experimental results show that the blue-ear disease NADC30-Like strain is widely spread in China and can cause clinical diseases of pigs.
In recent years, although scientists in various countries have conducted various researches on PRRSV, no method for effectively eradicating PRRS has been found yet, and prevention and control thereof depend on timely and accurate diagnosis and epidemic control. Diagnosis is based on that the antigenicity difference of PRRSV strains with different genotypes and pathogenicity of the control is large, the diagnosis technology and the control vaccine are different, and the diagnosis of different classification types according to the detection purpose is the primary example of the control of the disease. At present, the method adopted for detecting the porcine reproductive and respiratory syndrome virus mainly comprises the traditional virus separation and identification, ELISA method and common PCR detection. The virus separation operation is complicated, the time consumption is long, and the omission ratio is high; ELISA methods are relatively simple and rapid, but for samples with a large amount of trace or impurities, the specificity and sensitivity are low, which may cause missed diagnosis or misdiagnosis. PCR is a novel molecular biology technology, and since the birth, a large number of target fragments can be obtained by gene amplification in a short time due to high specificity and sensitivity, so that the PCR becomes one of the important means for detecting PRRSV at present. However, the common PCR still has the defects of complicated operation, long detection time, easy pollution and the like. Therefore, the current market is in urgent need of rapid, accurate, high-sensitivity and portable reagent products capable of detecting and distinguishing diagnosis in field operation.
Disclosure of Invention
Based on the above-mentioned needs in the art, the invention aims to provide a freeze-dried microchip and a kit for identifying porcine reproductive and respiratory syndrome virus (PRRS) and NADC30-Like strain, which can rapidly and effectively detect PRRS virus, and has high accuracy, specificity and sensitivity and good repeatability.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the freeze-dried microchip for identifying the classical strain of the porcine reproductive and respiratory syndrome virus and the NADC30-Like strain is characterized in that a fluorescent PCR reaction system for identifying the classical strain of the porcine reproductive and respiratory syndrome virus and the NADC30-Like strain is fixed on the microchip through freeze-drying;
the fluorescent PCR reaction system comprises: the following primers and probes:
porcine reproductive and respiratory syndrome virus classical strain upstream primer: 5 '-TGTTGTGACTTGAGCGTCGAT-3',
porcine reproductive and respiratory syndrome virus classical strain downstream primer: 5 '-CAAAAGGCCAGGAACCGTAA-3',
porcine reproductive and respiratory syndrome virus classical strain Taqman probe sequence: 5 '-TTTGTGATGCTCGTCAGG-3';
NADC30-Like strain upstream primer: 5 '-GCCTCGCTCAGAACTTCCT-3',
NADC30-Like strain downstream primer: 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, mg 2+ ；
Preferentially, the 5' -end of Taqman probes of the classical strain of the porcine reproductive and respiratory syndrome virus and the NADC30-Like strain are marked with fluorescent report groups; the 3' -end is marked with a fluorescence quenching group.
The fluorescent PCR reaction system comprises: upstream primer 0.4. Mu.M; 0.4. Mu.M of downstream primer; taqman probe 0.4. Mu.M; DNA polymerase 0.5U/. Mu.L; reverse transcriptase 0.5U/. Mu.L; dNTP 0.3mM; mg of 2+ 3mM; trehalose 5 μm; tris-Cl5mM, balance sterile deionized water, total volume of 36. Mu.L, 1.2. Mu.L per well volume;
preferentially, the fluorescence reporter group marked at the 5' end of the Taqman probe of the classical strain of the porcine reproductive and respiratory syndrome virus is a FAM fluorescence reporter group;
the fluorescence report group marked at the 5' -end of the Taqman probe of the porcine reproductive and respiratory syndrome virus NADC30-Like strain is a ROX fluorescence report group;
the Taqman probe 3' -end marked fluorescence quenching groups of the porcine reproductive and respiratory syndrome virus classical strain and the NADC30-Like strain are MGB quenching groups.
A plurality of sample adding holes are formed in the microchip; the fluorescent PCR reaction system is fixed in the sample adding hole through freeze drying;
preferably, the microchip on the sample application hole is 30; the structure of the microchip is matched with the structure of a sample adding plate of the PCR instrument.
The lyophilization comprises the following steps: placing the microchip provided with the fluorescent PCR reaction system at-80 ℃ for freezing for 1h, and then performing equipment freeze-drying;
preferably, the apparatus lyophilization comprises: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the retention time is 1h during pre-freezing, and then the equipment is vacuumized and kept for freeze drying for 1h; and in the desorption drying stage, the temperature of the separator is increased to-25 ℃ for 1h, then the temperature of the separator is increased to 37 ℃ and kept for 2h, and finally the temperature of the separator is reduced to 25 ℃ and kept for 1h.
The kit for identifying the classical strain of the porcine reproductive and respiratory syndrome virus and the NADC30-Like strain comprises the freeze-dried microchip.
The kit further comprises: diluting the 10 Xdiluent into 2 Xdiluent by using water without nuclease for dripping into a sample adding hole of the freeze-dried microchip, and then placing the freeze-dried microchip on a fluorescence PCR instrument for fluorescence PCR amplification.
The kit further comprises: mineral oil, sealing the sample application well on the lyophilized microchip;
positive control, specifically a mixture of classical strain of porcine reproductive and respiratory syndrome virus and genomic cDNA of NADC30-Like strain;
the negative control is in particular nuclease-free water.
The method for identifying the classical strain of the porcine reproductive and respiratory syndrome virus and the NADC30-Like strain is characterized in that the freeze-dried microchip and/or the kit is adopted to carry out fluorescent PCR amplification on a sample to be detected.
After a sample to be detected and a diluent are added into a sample adding hole of the freeze-dried microchip, the freeze-dried microchip is placed on a fluorescent PCR instrument for carrying out the fluorescent PCR amplification;
preferably, the dilution is 10 Xbuffer;
further preferably, the reaction procedure of the fluorescent PCR amplification is: 50 ℃ for 10min;95 ℃ for 1min; the fluorescent signal was detected at the end of extension of each cycle by performing 40 cycles with 1 cycle at 95℃for 5s and 60℃for 15 s.
The invention provides a fluorescent RT-PCR detection kit, which comprises a freeze-dried 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-dried microchip comprises a primer, a probe, taq enzyme, reverse transcriptase, trehalose and Tris-Cl, dNTP, mg 2+ 。
The PCR detection kit for the porcine reproductive and respiratory syndrome virus (PRRS) and NADC30-Like strain virus fluorescent freeze-dried microchip is characterized in that the 3' -end of the nucleotide sequence of a Taqman probe is marked with an MGB fluorescent quenching group. The freeze-dried microchip is coated with primers and Taqman probes having the following nucleotide sequences:
porcine reproductive and respiratory syndrome classical strain upstream primer: 5 '-TGTTGTGACTTGAGCGTCGAT-3',
porcine reproductive and respiratory syndrome classical strain downstream primer: 5 '-CAAAAGGCCAGGAACCGTAA-3',
Porcine reproductive and respiratory syndrome classical strain Taqman probe: 5 'FAM-TTTGTGATGCTCGTCAGG-MGB-3'.
NADC30-Like strain upstream primer: 5 '-GCCTCGCTCAGAACTTCCT-3',
NADC30-Like strain downstream primer: 5 '-CACCACGATGTAGGCTTCAG-3',
NADC30-Like strain Taqman probe: 5 'ROX-AGCATCTCGTCTCC-MGB-3'.
The final concentration of the freeze-drying system of the fluorescent quantitative PCR comprises: upstream primer 0.4. Mu.M; 0.4. Mu.M of downstream primer; taqman probe 0.4. Mu.M; DNA polymerase 0.5U/. Mu.L; reverse transcriptase 0.5U/. Mu.L; dNTP 0.3mM; mg of 2+ 3mM; trehalose 5 μm; tris-Cl 5mM; the balance of sterilized deionized water;
preferably, the total volume of the lyophilization system of the fluorescent quantitative PCR is 36 μl, and the volume per well is 1.2 μl;
the whole freeze-dried microchip sample loading holes of the AriaYSB freeze-dried microchip of the fluorescence quantitative PCR are 30, and 1.2 mu L of each microchip hole of the fluorescence PCR reaction system (comprising a porcine reproductive and respiratory syndrome strain and a NADC30-Like strain 2 sets of primer probes) is added.
The fluorescent quantitative PCR freeze-dried microchip was frozen for 1h at-80 ℃. The equipment freeze-drying conditions are as follows: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the retention time is 1h during pre-freezing, and then the equipment is vacuumized and kept for freeze drying for 1h; and in the desorption drying stage, the temperature of the separator is increased to-25 ℃ for 1h, then the temperature of the separator is increased to 37 ℃ and kept for 2h, and finally the temperature of the separator is reduced to 25 ℃ and kept for 1h.
The kit for detecting the fluorescence RT-PCR of the freeze-dried microchip of the porcine reproductive and respiratory syndrome classical strain and the NADC30-Like strain virus is characterized in that the reaction conditions of the fluorescence RT-PCR of the freeze-dried microchip are as follows: the temperature is 50 ℃ for 10min, and the cycle is the first step; 95 ℃ for 1min, which is the second circulation; 95℃for 5s and 60℃for 15s, the third step, 40 cycles, was performed, and fluorescence signal detection was performed at the end of extension of each cycle in the third step.
The kit for detecting the fluorescence RT-PCR of the freeze-dried microchip of the porcine reproductive and respiratory syndrome classical strain and the NADC30-Like strain virus comprises a PCR freeze-dried microchip, wherein the PCR freeze-dried microchip comprises a primer with the following nucleotide sequence and a Taqman probe: porcine reproductive and respiratory syndrome classical strain upstream primer: 5 '-TGTTGTGACTTGAGCGTCGAT-3', downstream primer of blue-ear classical strain: 5 '-CAAAAGGCCAGGAACCGTAA-3', porcine reproductive and respiratory syndrome classical strain Taqman probe: 5 'FAM-TTTGTGATGCTCGTCAGG-MGB-3'; NADC30-Like strain upstream primer: 5 '-GCCTCGCTCAGAACTTCCT-3', NADC30-Like strain downstream primer: 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 marked with an MGB fluorescence quenching group.
The 5' -end of the nucleotide sequence of the Taqman probe is marked with a FAM fluorescent reporter group; the NADC30-Like strain virus detects the ROX fluorescent reporter group marked at the 5' end of the nucleotide sequence of the Taqman probe.
The freeze-dried microchip reagent also comprises DNA polymerase, reverse transcriptase, dNTP and Mg 2+ Trehalose, tris-Cl.
The whole freeze-dried microchip loading holes of the freeze-dried AriaYSB microchip of the fluorescence quantitative PCR are 30, and the above fluorescence PCR reaction system (comprising the porcine reproductive and respiratory syndrome strain and the NADC30-Like strain 2 sets of primer probes) is diluted with 2 x diluent, and 1.2 μl is added to each microchip hole.
The freeze-drying condition is that the freeze-drying microchip of fluorescent quantitative PCR is frozen for 1h before the temperature of minus 80 ℃. The equipment freeze-drying conditions are as follows: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the retention time is 1h during pre-freezing, and then the equipment is vacuumized and kept for freeze drying for 1h; and in the desorption drying stage, the temperature of the separator is increased to-25 ℃ for 1h, then the temperature of the separator is increased to 37 ℃ and kept for 2h, and finally the temperature of the separator is reduced to 25 ℃ and kept for 1h.
The conventional reagents comprise commercial molecular reagents including mineral oil, diluent and water without nuclease;
the commercial diluent was 10 Xbuffer (without Mg 2+ ) The solution was diluted to 2 Xbuffer using nuclease-free water.
The 1.2 mu L reaction system of the freeze-dried microchip fluorescence quantitative PCR comprises: fluorescent quantitative PCR freeze-dried reagent hole and 0.6 mu L of diluent; sample RNA was detected at 0.6. Mu.L.
The kit also comprises a positive control, wherein the positive control is a mixture of porcine reproductive and respiratory syndrome classical strain and NADC30-Like strain virus genome cDNA.
The kit for detecting the porcine reproductive and respiratory syndrome virus (PRRS) by using the NADC30-Like strain virus freeze-dried microchip fluorescence RT-PCR is characterized in that the primer and/or the kit are adopted to detect a sample to be detected.
The detection refers to the fluorescent quantitative PCR detection of the freeze-dried microchip; the upstream primer is 0.4. Mu.M; 0.4. Mu.M of downstream primer; taqman probe 0.4. Mu.M; DNA polymerase 0.5U/. Mu.L; reverse transcriptase 0.5U/. Mu.L; dNTP 0.3mM; mg of 2+ 3mM; trehalose 5 μm; tris-Cl 5mM; the balance was sterile deionized water, in a total volume of 36 μl, 1.2 μl per well volume.
The reaction conditions of the freeze-dried microchip fluorescence RT-PCR include: the temperature is 50 ℃ for 10min, and the cycle is the first step; 95 ℃ for 1min, which is the second circulation; 95℃for 5s and 60℃for 15s, 40 cycles of the third step, with fluorescence signal detection at the end of extension of each cycle.
By adopting the technical scheme, the invention has at least the following advantages:
(1) According to the invention, primers are designed according to the domestic discovered Porcine Reproductive and Respiratory Syndrome (PRRS) classical strain and NADC30-Like strain virus NSP2 genes, specific primers and Taqman-MGB probes are synthesized, and the PRRS and NADC30-Like strain viruses can be rapidly and sensitively detected simultaneously by adopting a fluorescent quantitative PCR method, and the PRRS and NADC30-Like strain viruses are high in accuracy, specificity and sensitivity and good in repeatability.
(2) The invention adopts the high copy target gene on one hand and adopts the Taqman-MGB probe fluorescent quantitative PCR detection method on the other hand, so that the sensitivity of the fluorescent quantitative PCR detection by using the Taqman-MGB probe method is about 100 times that of the common PCR.
(3) The quantitative detection technology Taqman-MGB fluorescence quantitative PCR (Real-time PCR) is adopted, so that the method (Real-time PCR) has the advantages of pollution prevention due to single tube closed operation, high automation degree, strong specificity, real-time monitoring and the like, and effectively solves the limitation that the traditional method can only detect the end point.
(4) Compared with the currently used fluorescent PCR detection technology, the freeze-dried microchip technology has smaller reaction system of only 1.2 mu L and conventional reaction system of 20-25 mu L, so that the microchip technology can save RT-PCR amplification reagent and the dosage of samples.
(5) The smaller reaction system of the freeze-dried microchip can ensure that the system is heated more uniformly and the temperature rising and falling speed is faster in the PCR amplification process. The temperature rising rate (10-12 ℃/S) is faster than the temperature rising rate (3-5 ℃/S) of the currently adopted fluorescent PCR detection system. The whole fluorescent PCR process (including sample addition) can be completed within 40 minutes, and a computer automatically reports the result, so that electrophoresis and other subsequent work are not needed, the operation is convenient, and the pollution is reduced.
(6) The freeze-dried microchip can be stored and transported at normal temperature, so that repeated freeze thawing of the reagent is avoided, and the detection result is more stable.
The invention adopts a Real-time fluorescence quantitative PCR technology (Real-time PCR), which is a method of adding a fluorescent group into a PCR reaction system, monitoring the whole PCR process in Real time by utilizing fluorescent signal accumulation and finally quantitatively analyzing an unknown template through a standard curve.
The microchip used in the present invention is a reaction region formed of a silica gel or an aluminum plate with a microreactor (the volume and mass of the microreactor depend on the type of microchip) covered with a protective film. The reaction area of the microchip is covered with a layer of mineral oil. Reagents are injected into the microreactor through a layer of mineral oil by a manual or automatic pipette with a gun head, so that cross contamination of detection samples and evaporation of a reaction system can be avoided.
The PCR reaction reagent is freeze-dried on a microchip, and the Taqman probe fluorescent quantitative PCR technology is combined, so that the nucleic acid is analyzed by simultaneously reading fluorescent signals generated by PCR products on each reactor during thermal cycling of the microchip. The microchip technology adopted by the invention is different from the PCR amplification carried by the plastic PCR tube which is commonly used at present as a carrier, but adopts a metal carrier microchip, and the reaction system and the sample are directly added on the metal carrier for PCR amplification. The heat conduction efficiency and the temperature rise and drop rate of the metal carrier are faster, and the reaction procedure 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 preservation of reaction reagents, capability of quantitative detection, small pollution possibility of microchip operation and the like. The product of the invention can rapidly identify and distinguish porcine reproductive and respiratory syndrome classical strain and NADC30-Like strain viruses under the condition of consistent reaction conditions, has simple operation, and can be completed within 1 hour from sample processing to result analysis.
In conclusion, by adopting the technical scheme, the microchip fluorescence quantitative PCR reaction system capable of rapidly and effectively detecting the porcine reproductive and respiratory syndrome classical strain and the NADC30-Like strain virus is developed by designing the specific primers and the probes and optimizing the microchip freeze-drying condition, and the detection kit based on the method is prepared. 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 10TCID 50 The sensitivity is very high, the variation coefficient in the detection kit is between 0.91 and 1.08 percent, the variation coefficient between batches is between 1.13 and 1.87 percent, and the repeatability is good.
Drawings
FIG. 1 is a specific assay for the detection of porcine reproductive and respiratory syndrome virus by fluorescence RT-PCR of a lyophilized microchip. In the figure, 1: porcine reproductive and respiratory syndrome classical strain virus; 2: highly pathogenic classical variants; 3: NADC30-Like strain; 4: porcine transmissible gastroenteritis virus; 5: epidemic diarrhea virus; 6: rotavirus; 7: swine influenza virus; 8: swine fever virus; 9: a pig ring; 10: the pigs are tiny; 11: porcine pseudorabies nucleic acid; 12: nuclease-free water.
FIG. 2 is a specific assay for detecting porcine reproductive and respiratory syndrome NADC30-Like strain virus by fluorescence RT-PCR of a lyophilized microchip. In the figure, 1: NADC30-Like strain; 2: highly pathogenic classical variants; 3: porcine reproductive and respiratory syndrome classical strain virus; 4: porcine transmissible gastroenteritis virus; 5: epidemic diarrhea virus; 6: rotavirus; 7: swine influenza virus; 8: swine fever virus; 9: a pig ring; 10: the pigs are tiny; 11: porcine pseudorabies nucleic acid; 12: nuclease-free water.
FIG. 3 shows the result of detecting sensitivity of porcine reproductive and respiratory syndrome classical strain virus by using a freeze-dried microchip fluorescent RT-PCR reagent. In the figure, a: 1X 10 6 TCID 50 /mL；B：1×10 5 TCID 50 /mL；C：1×10 4 TCID 50 /mL；D：1×10 3 TCID 50 /mL；E：1×10 2 TCID 50 /mL；F：1×10 1 TCID 50 /mL；G:1×10 0 TCID 50 /mL; h, no nuclease water.
FIG. 4 shows the result of detecting porcine reproductive and respiratory syndrome classical strain virus sensitivity by using a conventional fluorescent RT-PCR reagent. In the figure, a: 1X 10 6 TCID 50 /mL；B：1×10 5 TCID 50 /mL；C：1×10 4 TCID 50 /mL；D：1×10 3 TCID 50 /mL；E：1×10 2 TCID 50 /mL；F：1×10 1 TCID 50 /mL；G:1×10 0 TCID 50 /mL; h, no nuclease water.
FIG. 5 shows the result of the standard curve of the detection of porcine reproductive and respiratory syndrome virus by using the fluorescence RT-PCR reagent of the freeze-dried microchip.
FIG. 6 is a standard curve of the detection of porcine reproductive and respiratory syndrome virus by using a conventional fluorescent RT-PCR reagent.
Detailed Description
In order to make the technical means of the present invention more clearly understood, the present invention will be described in further detail below with reference to the accompanying drawings and the detailed description. Unless otherwise indicated, the reagents used in the following examples were all analytical grade reagents and were commercially available from regular sources.
Sources or documentations of biological materials
The porcine reproductive and respiratory syndrome virus classical strain (VR-2322 strain) and the NADC30-Like strain used in the experimental example are standard inactivated strains for animal epidemic control in China, and can be obtained commercially. Other strains are also commercially available.
Group 1 examples: the freeze-dried microchip of the present invention
The embodiment of the group provides a freeze-dried microchip for identifying a classical strain of porcine reproductive and respiratory syndrome virus and a NADC30-Like strain, which is characterized in that a fluorescent PCR reaction system for identifying the classical strain of porcine reproductive and respiratory syndrome virus and the NADC30-Like strain is fixed on the microchip through freeze-drying;
The fluorescent PCR reaction system comprises: the following primers and probes:
porcine reproductive and respiratory syndrome virus classical strain upstream primer: 5 '-TGTTGTGACTTGAGCGTCGAT-3',
porcine reproductive and respiratory syndrome virus classical strain downstream primer: 5 '-CAAAAGGCCAGGAACCGTAA-3',
porcine reproductive and respiratory syndrome virus classical strain Taqman probe: 5 '-TTTGTGATGCTCGTCAGG-3';
NADC30-Like strain upstream primer: 5 '-GCCTCGCTCAGAACTTCCT-3',
NADC30-Like strain downstream primer: 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, mg 2+ ；
Preferentially, the 5' -end of Taqman probes of the classical strain of the porcine reproductive and respiratory syndrome virus and the NADC30-Like strain are marked with fluorescent report groups; 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; 0.4. Mu.M of downstream primer; taqman probe 0.4. Mu.M; DNA polymerase 0.5U/. Mu.L; reverse transcriptase 0.5U/. Mu.L; dNTP 0.3mM; mg of 2+ 3mM; trehalose 5 μm; tris-Cl 5mM, the balance of sterilized deionized water;
the final concentrations of these components were not only those of the fluorescent PCR reaction system before lyophilization, but also those after lyophilization, after 0.6uL and 0.6uL of 2 Xdilution were added.
The total volume of the fluorescent PCR reaction system formulated prior to lyophilization is typically 36. Mu.L, with a 1.2. Mu.L per well volume.
Preferentially, the fluorescence reporter group marked at the 5' end of the Taqman probe of the classical strain of the porcine reproductive and respiratory syndrome virus is a FAM fluorescence reporter group;
the fluorescence report group marked at the 5' -end of the Taqman probe of the porcine reproductive and respiratory syndrome virus NADC30-Like strain is a ROX fluorescence report group;
the Taqman probe 3' -end marked fluorescence quenching groups of the porcine reproductive and respiratory syndrome virus classical strain and the NADC30-Like strain are MGB quenching groups.
In some embodiments, the microchip is provided with a plurality of sample application holes; the fluorescent PCR reaction system is fixed in the sample adding hole through freeze drying;
preferably, the microchip on the sample application hole is 30; the structure of the microchip is matched with the structure of a sample adding plate of the PCR instrument. The microchip has a structure similar to that of a PCR plate, but the microchip of the invention is uncovered, mineral oil is required to seal the sample adding hole, and the hole is not easy to be crossed during sample adding and is not easy to pollute. Therefore, the phenomenon of overflow of the PCR system flow or cross hole pollution can not occur.
In other embodiments, the lyophilization comprises the steps of: placing the microchip provided with the fluorescent PCR reaction system at-80 ℃ for freezing for 1h, and then performing equipment freeze-drying;
The microchip provided with the fluorescent PCR reaction system is firstly frozen at the temperature of minus 80 ℃ for 1h to be pre-frozen, 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 this step.
Device lyophilization refers to lyophilization in a specialized lyophilization apparatus by setting the operating conditions of the apparatus; preferably, the lyophilization apparatus refers to a vacuum freeze dryer.
Preferably, the apparatus lyophilization comprises: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the retention time is 1h during pre-freezing, and then the equipment is vacuumized and kept for freeze drying for 1h; and in the desorption drying stage, the temperature of the separator is increased to-25 ℃ for 1h, then the temperature of the separator is increased to 37 ℃ and kept for 2h, and finally the temperature of the separator is reduced to 25 ℃ and kept for 1h.
The temperature of the partition plate refers to the temperature of a tray partition plate in a freeze dryer, and a microchip provided with the fluorescent PCR reaction system needs to be placed on the tray partition plate; the pre-freezing stage has the function of keeping the fluorescent PCR reaction system in a solid state. The main purpose of the step of vacuumizing the equipment and keeping the freeze drying for 1h is a vacuum freeze dryer, and the internal air is required to be pumped out to sublimate the water in the solid, so that the freeze drying effect is achieved. The next 2 steps, raising the temperature of the separator to-25 ℃ for 1h, and raising the temperature of the separator to 37 ℃ and for 2h, are to maintain the dry sublimation process; finally, the function of 'cooling the separator to 25 ℃ and keeping for 1 h' is to keep the freeze-dried finished product stable at 25 ℃, so that the whole freeze-drying process is basically completed.
The equipment freeze-drying process is a set of freeze-drying process which is created by the invention aiming at the microchip provided with the fluorescent PCR reaction system. Because the eutectic points of the freeze-drying reagents are different, the measurement is needed, and the heating time in the drying process and the drying time in the drying time are needed to be optimized. The effect of the components within the lyophilized reagent on the eutectic point requires adjustment of reagent component concentrations to be explored. In particular, the enzyme in the reaction system is placed at normal temperature for a long time, the activity of the enzyme is reduced, and a protective agent and a stabilizer component, such as trehalose 5 mu M, are required to be added; the Tris-Cl 5mM has the concentration which is verified and regulated by experiments to ensure 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-dried microchip is used for carrying out fluorescent PCR detection. In addition, the re-dissolution effect of the diluent used after the freeze-drying process is also required to be simultaneously considered, and the excellent effects of high sensitivity, high repeatability, high accuracy, high specificity and the like of the finally obtained detection method of the invention can be achieved by the freeze-dried microchip only through adjusting the freeze-drying process.
The person skilled in the art can synthesize the primers manually according to the disclosure of the invention, and use them for qualitatively or quantitatively detecting porcine reproductive and respiratory syndrome virus and NADC30-Like strain virus, so that the expected effect according to the invention can be obtained, and therefore any primers synthesized based on commercial purposes are put into a commercial package marked with the purpose of detecting porcine reproductive and respiratory syndrome virus and NADC30-Like strain virus, or the primers adopting the sequences are used for commercial detection of porcine reproductive and respiratory syndrome virus and NADC30-Like strain virus, which falls into the scope of the invention.
Group 2 examples: the kit of the invention
The present set of examples provides a kit for identifying classical strains of porcine reproductive and respiratory syndrome virus from NADC30-Like strains, comprising a lyophilized microchip according to any one of the embodiment of set 1.
In a further embodiment, the kit further comprises: and (3) dripping the diluent into a sample adding hole of the freeze-dried microchip, and then placing the freeze-dried microchip on a fluorescent PCR instrument for fluorescent PCR amplification.
The dilution is specifically PCR 10 Xbuffer, and the specific components do not contain Mg 2+ This dilution is commercially available containing 500mM KCl,100mM Tris-HCl,0.1% gelatin. In use, the dilution is diluted to 2 Xbuffer using nuclease-free water, and 0.6. Mu.L of the solution is added to each well of the microchip.
In a further embodiment, the kit further comprises: mineral oil is used for sealing the sample adding hole on the microchip, and the system is very trace, so that the sample is not easy to pollute due to the fact that the oil is not sealed, the air drying is easy, and the oil sealing is equivalent to a tube cover for covering the PCR tube.
Positive control, specifically a mixture of classical strain of porcine reproductive and respiratory syndrome virus and genomic cDNA of NADC30-Like strain;
the negative control is in particular nuclease-free water.
In specific embodiments, the 3 'end of the nucleotide sequence of the probe is labeled with an MGB quencher, and the 5' end is labeled with a FAM or ROX fluorescent reporter. The "MGB", "FAM" and "ROX" groups described above are all common fluorescent groups in the art, and other fluorescent quenching groups and fluorescent reporting groups common in the art may be selected by those skilled in the art to replace the "MGB", "FAM" and "ROX" herein, for example, common fluorescent quenching groups further include: BHQ-1, BHQ-2, dabcyl2; common fluorescent reporter groups may also be selected from: TET, HEX, 5-TAMRA, texas Red-X, cy (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 detection.
The freeze-dried microchip reagent also comprises DNA polymerase, reverse transcriptase, dNTP and Mg 2+ Trehalose, tris-Cl.
In a preferred embodiment, the conventional reagents include DNA polymerase, reverse transcriptase, dNTPs, mg 2+ Trehalose, tris-Cl; and/or commercial molecular agents including the mineral oil, diluent, nuclease-free water;
in a further preferred embodiment, the commercial diluent is 10 Xbuffer (without Mg 2+ ) The solution was diluted to 2 Xbuffer using nuclease-free water. There are also various DNA polymerases, reverse transcriptases, dNTPs, 10 Xbuffers commercially available in the art, and other brands and types of molecular reagents may be selected by those skilled in the art.
Group 3 examples: the detection method of the invention
The embodiment of the group provides a freeze-dried microchip fluorescence RT-PCR detection method for identifying Porcine Reproductive and Respiratory Syndrome (PRRS) classical strains and NADC30-Like strains. All embodiments of this group have the following features: the method comprises the following steps: the kit according to any one of the embodiments of group 1 is used for fluorescent PCR amplification of a sample to be tested.
In some embodiments, the freeze-dried microchip is placed on a fluorescent PCR instrument for the fluorescent PCR amplification after the sample to be tested and the diluent are added to the sample adding hole of the freeze-dried microchip;
Preferably, the diluent 2 Xbuffer is added in an amount of 0.6. Mu.L, and the positive control, negative control or sample is added in an amount of 0.6. Mu.L;
further preferably, the reaction procedure of the fluorescent PCR amplification is: 50 ℃ for 10min;95 ℃ for 1min; the fluorescent signal was detected at the end of extension of each cycle by performing 40 cycles with 1 cycle at 95℃for 5s and 60℃for 15 s.
In some embodiments, the detection is a fluorescent quantitative PCR detection; the freeze-dried microchip of the porcine reproductive and respiratory syndrome classical strain and the NADC30-Like strain comprises: upstream primer 0.4. Mu.M; 0.4. Mu.M of downstream primer; taqman probe 0.4. Mu.M; DNA polymerase 0.5U/. Mu.L; reverse transcriptase 0.5U/. Mu.L; dNTP 0.3mM; mg of 2+ 3mM; trehalose 5 μm; tris-Cl 5mM; the balance was sterile deionized water, in a total volume of 36 μl, 1.2 μl per well volume.
The number of the whole freeze-dried microchip sample loading holes of the freeze-dried microchip of the fluorescence quantitative PCR is 30, and 1.2 mu L of the fluorescence quantitative PCR system (comprising a porcine reproductive and respiratory syndrome strain and a NADC30-Like strain 2 primer probe) is added into each hole.
In some embodiments, the lyophilization conditions are fluorescent quantitative PCR of the lyophilized microchip before-80 ℃ frozen for 1h. The equipment freeze-drying conditions are as follows: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the retention time is 1h during pre-freezing, and then the equipment is vacuumized and kept for freeze drying for 1h; and in the desorption drying stage, the temperature of the separator is increased to-25 ℃ for 1h, then the temperature of the separator is increased to 37 ℃ and kept for 2h, and finally the temperature of the separator is reduced to 25 ℃ and kept for 1h.
In a further embodiment, the reaction procedure of fluorescent quantitative PCR comprises: the temperature is 50 ℃ for 10min, and the cycle is the first step; 95 ℃ for 1min, which is the second circulation; 95℃for 5s and 60℃for 15s, 40 cycles of the third step, with fluorescence signal detection at the end of extension of each cycle.
Experimental example 1 blue-ear classical strain and NADC30-Like strain virus freeze-dried microchip fluorescent RT-PCR detection kit
Specificity verification
1. Design of primers and Taqman-MGB probes
According to domestic detection of porcine reproductive and respiratory syndrome virus and NADC30-Like strain virus, specific conserved sequences of porcine reproductive and respiratory syndrome virus NSP2 genes of the NADC30-Like strain virus are found, and a plurality of pairs of primers and probes are designed. And finally, respectively determining a group of optimal primers and a Taqman-MGB probe through comparison and screening.
Porcine reproductive and respiratory syndrome classical strain upstream primer: 5' -TGTTGTGACTTGAGCGTCGAT-3
Porcine reproductive and respiratory syndrome classical strain downstream primer: 5' -CAAAAGGCCAGGAACCGTAA-3
Porcine reproductive and respiratory syndrome classical strain Taqman probe: 5 'FAM-TTTGTGATGCTCGTCAGG-MGB-3';
NADC30-Like strain upstream primer: 5' -GCCTCGCTCAGAACTTCCT-3
NADC30-Like strain downstream primer: 5' -CACCACGATGTAGGCTTCAG-3
NADC30-Like strain Taqman probe: 5 'ROX-AGCATCTCGTCTCC-MGB-3'.
Wherein the 5' end of the nucleotide sequence of the Taqman probe of the porcine reproductive and respiratory syndrome classical strain is marked with a FAM fluorescent reporter group; the 5' end of the nucleotide sequence of the Taqman probe of the NADC30-Like strain is marked with a ROX fluorescent reporter group. The 3' end-labeled MGB quenches the fluorophore. The reason for the quenching of the fluorophore to select MGB is that the TaqMan-MGB probe has the following advantages compared with the conventional TaqMan-TAMRA probe: (1) Increasing the TM value-15 bases on average increases 18 ℃, which can shorten the length of the probe, especially greatly facilitates the design of sequences with high AT content, and increases the TM value difference between paired and unpaired templates. (2) The signal to noise ratio is improved-because the quenching group at the 3' end of the probe is a fluorescent group which does not emit light, and the position of the quenching group and the reporter group in space is closer, the experimental result is more accurate, and the resolution ratio is higher.
2. Viral RNA extraction
The genome RNA of the highly pathogenic classical variant, NADC30-Like strain, porcine reproductive and respiratory syndrome classical strain virus, transmissible gastroenteritis virus, epidemic diarrhea virus, rotavirus, swine influenza virus and swine fever virus is extracted by using a total RNA extraction kit (EAR 002, beijing Yisen Biotechnology Co., ltd.). Porcine circus and porcine parvos and porcine pseudorabies genomic DNA were extracted using a total DNA extraction kit (Beijing Yisen Biotechnology Co., ltd., cat# EAD 001). Placing at-20deg.C for use.
3. Freeze-dried microchip preparation
The preparation of the freeze-dried microchip RT-PCR system is carried out according to the following reaction system:
the whole lyophilized microchip of the AriaYSB microchip (Beijing Yisen Biotechnology Co., ltd.) for fluorescence quantitative PCR had 30 wells, and 1.2. Mu.L of the above fluorescence quantitative PCR system (comprising porcine reproductive and respiratory syndrome strain and NADC30-Like strain 2 sets of primer probes) was added to each well.
The microchip coated with the PCR reagent was frozen for 1 hour at-80 ℃. The equipment freeze-drying conditions are as follows: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the retention time is 1h during pre-freezing, and then the equipment is vacuumized and kept for freeze drying for 1h; and in the desorption drying stage, the temperature of the separator is increased to-25 ℃ for 1h, then the temperature of the separator is increased to 37 ℃ and kept for 2h, and finally the temperature of the separator is reduced to 25 ℃ and kept for 1h.
4. Lyophilized microchip specificity verification
After adding 24. Mu.L nuclease-free water to 10 Xbuffer dilution and shaking and mixing, 0.6. Mu.L mineral oil was added to each well, and 600. Mu.L mineral oil was added to the microchip surface, after which all wells were covered. Then 0.6 mu L of high-pathogenicity classical variant, NADC30-Like strain, porcine reproductive and respiratory syndrome virus, porcine transmissible gastroenteritis virus, epidemic diarrhea virus, rotavirus, swine influenza 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 to the last well as a negative control.
The conditions for the fluorescent RT-PCR reaction of the lyophilized microchip were as follows: the temperature is 50 ℃ for 10min, and the cycle is the first step; 95 ℃ for 1min, which is the second circulation; 95℃for 5s and 60℃for 15s, 40 cycles of the third step, with fluorescence signal detection at the end of extension of each cycle.
The fluorescence reporter groups marked by the probes are 2 types, namely FAM fluorescence reporter groups and ROX fluorescence reporter groups, and when the fluorescence PCR software is used for analyzing the results, the corresponding fluorescence reporter groups can be respectively selected to display the results, for example, the FAM fluorescence reporter group probes, namely probes of classical strain of porcine reproductive and respiratory syndrome virus, only display the detection results of the primer probes of classical strain of porcine reproductive and respiratory syndrome virus. The ROX probe (channel) is the probe of the porcine reproductive and respiratory syndrome virus NADC30-Like strain, and only shows the detection result of the set of primer probes of the porcine reproductive and respiratory syndrome virus NADC30-Like strain. The results for 2 fluorescent reporter groups can be shown separately.
The experimental results are shown in the figure, the freeze-dried microchip is coated with a double fluorescent RT-PCR reagent of a porcine reproductive and respiratory syndrome strain and a NADC30-Like strain, and the results in the figure 1 show that the porcine reproductive and respiratory syndrome strain virus RNA detected by a porcine reproductive and respiratory syndrome strain virus channel is amplified positively, and the Ct value is 22.93, and an amplification curve exists. The high pathogenic classical variant, NADC30-Like strain, transmissible gastroenteritis virus, epidemic diarrhea virus, rotavirus, swine influenza virus, swine fever virus, porcine circus, porcine parvovirus, porcine pseudorabies and negative control sample have no non-specific amplification. The result of FIG. 2 shows that NADC30-Like strain channel detects NADC30-Like strain RNA as positive amplification, ct value is 24.09, and amplification curve is provided. The high pathogenic classical variant, porcine reproductive and respiratory syndrome virus, porcine transmissible gastroenteritis virus, epidemic diarrhea virus, rotavirus, swine influenza virus, swine fever virus, porcine circus, porcine parvovirus, porcine pseudorabies and negative control sample have no non-specific amplification. The results obtained are completely identical to the expected ones. As can be seen from the amplification curves of all samples, the curve coincidence is good in the early stage of amplification, especially around the fluorescence threshold (threshold).
Experimental example 2, porcine reproductive and respiratory syndrome classical strain and NADC30-Like strain virus freeze-dried microchip fluorescent RT-PCR detection kit
Sensitivity verification and comparison with conventional fluorescent RT-PCR reagents
The concentration was 1X 10 7 TCID 50 A10-fold gradient dilution was performed on/mL of porcine reproductive and respiratory syndrome virus solution. Extraction of 1X 10 6 TCID 50 /mL～1×10 0 TCID 50 And (3) carrying out universal sensitivity detection on the porcine reproductive and respiratory syndrome classical strain viruses by using the freeze-dried microchip fluorescent RT-PCR reagent and the conventional fluorescent RT-PCR reagent by taking the genome RNA of the porcine reproductive and respiratory syndrome classical strain viruses with each gradient concentration as a template and the water without the nuclease as negative control.
A lyophilized microchip fluorescent RT-PCR system was prepared as in example 1, and after adding 24. Mu.L nuclease-free water to a 10 Xbuffer dilution and mixing well, eachThe wells were added with 0.6. Mu.L and 600. Mu.L of mineral oil was added to the microchip surface, after which it covered all of the wells. 1X 10 of each of the above-mentioned substances was added to the wells 6 TCID 50 /mL～1×10 0 TCID 50 Each gradient concentration of porcine reproductive and respiratory syndrome virus genomic RNA was/mL and 0.6. Mu.L of nuclease-free water was added to the final 1 well as a negative control.
The conditions for the fluorescent RT-PCR reaction of the lyophilized microchip were as follows: the temperature is 50 ℃ for 10min, and the cycle is the first step; 95 ℃ for 1min, which is the second circulation; 95℃for 5s and 60℃for 15s, 40 cycles of the third step, with fluorescence signal detection at the end of extension of each cycle.
Preparing a conventional fluorescent RT-PCR reagent system:
conventional fluorescent RT-PCR was as follows: the temperature is 50 ℃ for 30min, and the cycle is the first step; the temperature is 95 ℃ for 5min, and the second step of circulation is performed; 95℃15s,60℃45s, 40 cycles of the third step, each cycle of which was extended to the end, were subjected to fluorescent signal detection.
FIG. 3 shows that the concentration of the lowest detection sample is 1X 10 by using the freeze-dried microchip fluorescence RT-PCR 1 TCID 50 The Ct value of the reaction cycle number 40 can greatly meet the minimum detection requirement because of the concentration of the catalyst per mL and Ct value of 33.97. As can be seen from the amplification curves of the starting templates at different concentrations, the curve has a flat base line, a distinct exponential region and a larger slope, which indicates that the amplification of the templates is more desirable under these conditions. FIG. 4 shows that the concentration of the sample for the lowest detection of the conventional fluorescent RT-PCR is 1×10 2 TCID 50 Ct value was 34.57 per mL. The results show that the sensitivity of the freeze-dried microchip fluorescent RT-PCR detection is higher (about 10 times higher) than that of the conventional fluorescent RT-PCR detection. Because the Ct value and the logarithm of the initial template have a linear relation, a standard curve can be manufactured by using the Ct value and the actual concentration of the template, and a linear relation equation is listed. The results of FIGS. 5 and 6 show that the fluorescence RT-PCR amplification efficiency of the freeze-dried microchip is 105.40%, the fluorescence RT-PCR amplification efficiency of the conventional fluorescence RT-PCR amplification efficiency is 87.58%, and the fluorescence RT-PCR amplification efficiency of the freeze-dried microchip is higher. Both of which are formed by a single-layer process The standard curve formula and the correlation coefficient are relatively close. The correlation coefficient of the fluorescence RT-PCR of the freeze-dried microchip and the conventional fluorescence RT-PCR is 0.99, so the correlation of the fluorescence RT-PCR of the freeze-dried microchip and the conventional fluorescence RT-PCR is good. The fluorescent RT-PCR reaction system of the freeze-dried 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-dried microchip is reduced by more than 20 times. The running time of the freeze-dried microchip fluorescent RT-PCR reaction is less than 40 minutes, the conventional fluorescent RT-PCR reaction time is 120 minutes, and the running time is shortened by 2/3.
Experimental example 3: porcine reproductive and respiratory syndrome classical strain and NADC30-Like strain virus freeze-dried microchip fluorescent RT-PCR detection kit
Preparation and detection of (3)
1. Preparation of the kit:
preparation of lyophilized microchip:
the preparation of the freeze-dried microchip RT-PCR system is carried out according to the following reaction system:
the whole lyophilized microchip loading well of the AriaYSB microchip (beijing glossobao biotechnology limited) for fluorescent quantitative PCR was 30, and 1.2 μl of each microchip well was added to the above fluorescent quantitative PCR system (comprising porcine reproductive and respiratory syndrome strain and NADC30-Like strain 2 sets of primer probes).
The microchip coated with the PCR reagent was frozen for 1 hour at-80 ℃. The equipment freeze-drying conditions are as follows: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the retention time is 1h during pre-freezing, and then the equipment is vacuumized and kept for freeze drying for 1h; and in the desorption drying stage, the temperature of the separator is increased to-25 ℃ for 1h, then the temperature of the separator is increased to 37 ℃ and kept for 2h, and finally the temperature of the separator is reduced to 25 ℃ and kept for 1h.
Reagent 1: dilution Taq Buffer (10×) (Thermo Scientific, cat# B650060), 6 μl;
reagent 2: mineral oil (Sangon Biotech, cat. A630217) 1mL;
reagent 3: 30 mu L of positive control (NADC 30-Like strain, porcine reproductive and respiratory syndrome classical strain virus genome cDNA mixture);
reagent 4: negative control (no nuclease) 30 μl;
reagent 5: nuclease-free water 50 μl.
2. Repetitive analysis of kits
3 samples of known positives were selected for each of the intra-batch and inter-batch replicates. Repeated detection in batch: 3 known positive samples were run in the same batch of experiments, with 3 replicates per sample. Experiments were repeated between batches: 3 known positive samples were tested in batches, each sample was tested separately, and each sample was set up with 3 replicates.
The fluorescence RT-PCR reaction system of each freeze-dried microchip is 1.2 mu L: before using the reagent 1, 24 mu L of the reagent 5 is required to be sucked, added into the reagent 1, and mixed uniformly by shaking, and then 0.6 mu L of the reagent is added into each hole. 600. Mu.L of reagent 2 was added to the microchip surface, after which it covered all of the wells. Each well was added with 0.6. Mu.L of each of the positive sample, reagent 3 (positive control) or reagent 4 (negative control).
The conditions for the fluorescent RT-PCR reaction of the lyophilized microchip were as follows: the temperature is 50 ℃ for 10min, and the cycle is the first step; 95 ℃ for 1min, which is the second circulation; 95℃for 5s and 60℃for 15s, 40 cycles of the third step, which was followed by fluorescence signal detection at the end of extension of each cycle, and recording of the experimental results.
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 reproducibility assay
By adopting the technical scheme, the microchip fluorescence quantitative PCR detection reaction system capable of rapidly and effectively detecting the porcine reproductive and respiratory syndrome and NADC30-Like strain viruses is developed by designing the specific primers and probes and optimizing the microchip freeze-drying condition, and meanwhile, the detection kit based on the method is prepared, the detection method is high in accuracy, specificity and sensitivity, and the detection kit is good in repeatability.
The above description is only of the preferred embodiments of the present invention, and is not intended to limit the invention in any way, and some simple modifications, equivalent variations or modifications can be made by those skilled in the art using the teachings disclosed herein, which fall within the scope of the present invention.
SEQUENCE LISTING
<110> Beijing Yisen Bao biotechnology Co., ltd
China Animal Disease Control Center
<120> lyophilized microchip, kit and method for identifying classical strain of porcine reproductive and respiratory syndrome virus and NADC30-Like strain
<130> P180494/YSB
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> classical strain upstream primer of porcine reproductive and respiratory syndrome virus
<400> 1
tgttgtgact tgagcgtcga t 21
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> porcine reproductive and respiratory syndrome virus classical strain downstream primer
<400> 2
caaaaggcca ggaaccgtaa 20
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Taqman probe sequence of classical strain of porcine reproductive and respiratory syndrome virus
<400> 3
tttgtgatgc tcgtcagg 18
<210> 4
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> NADC30-Like strain upstream primer
<400> 4
gcctcgctca gaacttcct 19
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> NADC30-Like strain downstream primer
<400> 5
caccacgatg taggcttcag 20
<210> 6
<211> 14
<212> DNA
<213> Artificial Sequence
<220>
<223> NADC30-Like strain Taqman probe sequence
<400> 6
agcatctcgt ctcc 14
Claims (17)
1. The freeze-dried microchip for identifying the classical strain of the porcine reproductive and respiratory syndrome virus and the NADC30-Like strain is characterized in that a fluorescent PCR reaction system for identifying the classical strain of the porcine reproductive and respiratory syndrome virus and the NADC30-Like strain is fixed on the microchip through freeze-drying;
the fluorescent PCR reaction system comprises: the following primers and probes:
porcine reproductive and respiratory syndrome virus classical strain upstream primer: 5 '-TGTTGTGACTTGAGCGTCGAT-3',
porcine reproductive and respiratory syndrome virus classical strain downstream primer: 5 '-CAAAAGGCCAGGAACCGTAA-3',
porcine reproductive and respiratory syndrome virus classical strain Taqman probe sequence: 5 '-TTTGTGATGCTCGTCAGG-3';
NADC30-Like strain upstream primer: 5 '-GCCTCGCTCAGAACTTCCT-3',
NADC30-Like strain downstream primer: 5 '-CACCACGATGTAGGCTTCAG-3',
NADC30-Like strain Taqman probe sequence: 5 '-AGCATCTCGTCTCC-3';
the minimum detection sample concentration of the freeze-dried microchip is 1 multiplied by 10 1 TCID 50 /mL。
2. The lyophilized microchip according to claim 1, wherein the fluorescent PCR reaction system further comprises: taq enzyme, reverse transcriptase, trehalose, tris-Cl, dNTP, mg 2+ 。
3. The lyophilized microchip according to claim 1, wherein the 5' end of the Taqman probes of classical strain of porcine reproductive and respiratory syndrome virus and NADC30-Like strain is labeled with a fluorescent reporter group; the 3' -end is marked with a fluorescence quenching group.
4. The lyophilized microchip according to claim 1, wherein the fluorescent PCR reaction system comprises: upstream primer 0.4. Mu.M; 0.4. Mu.M of downstream primer; taqman probe 0.4. Mu.M; DNA polymerase 0.5U/. Mu.L; reverse transcriptase 0.5U/. Mu.L; dNTP0.3mM; mg of 2+ 3mM; trehalose 5 μm; tris-Cl5mM, balance sterile deionized water, total volume 36. Mu.L, 1.2. Mu.L per well volume.
5. The lyophilized microchip according to claim 2, wherein the fluorescent PCR reaction system comprises: upstream primer 0.4. Mu.M; 0.4. Mu.M of downstream primer; taqman probe 0.4. Mu.M; DNA polymerase 0.5U/. Mu.L; reverse transcriptase 0.5U/. Mu.L; dNTP0.3mM; mg of 2+ 3mM; trehalose 5 μm; tris-Cl5mM, balance sterile deionized water, total volume 36. Mu.L per wellThe volume was 1.2. Mu.L.
6. The lyophilized microchip according to claim 3, wherein the fluorescent reporter group labeled at the 5' end of the Taqman probe of the classical strain of porcine reproductive and respiratory syndrome virus is a FAM fluorescent reporter group;
The fluorescence report group marked at the 5' -end of the Taqman probe of the porcine reproductive and respiratory syndrome virus NADC30-Like strain is a ROX fluorescence report group;
the Taqman probe 3' -end marked fluorescence quenching groups of the porcine reproductive and respiratory syndrome virus classical strain and the NADC30-Like strain are MGB quenching groups.
7. The lyophilized microchip according to any one of claims 1-6, wherein a plurality of sample application wells are provided on the microchip; the fluorescent PCR reaction system was immobilized within the loading well by lyophilization.
8. The lyophilized microchip according to claim 7, wherein the number of loading wells on the microchip is 30; the structure of the microchip is matched with the structure of a sample adding plate of the PCR instrument.
9. The lyophilized microchip according to any one of claims 1-6, wherein the lyophilization comprises the steps of: and (3) placing the microchip provided with the fluorescent PCR reaction system at the temperature of-80 ℃ for freezing for 1h, and then performing equipment freeze-drying.
10. The lyophilized microchip according to claim 9, wherein the device lyophilization comprises: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the retention time is 1h during pre-freezing, and then the equipment is vacuumized and kept for freeze drying for 1h; and in the desorption drying stage, the temperature of the separator is increased to-25 ℃ for 1h, then the temperature of the separator is increased to 37 ℃ and kept for 2h, and finally the temperature of the separator is reduced to 25 ℃ and kept for 1h.
11. A kit for identifying classical strains of porcine reproductive and respiratory syndrome virus from NADC30-Like strains, comprising a lyophilized microchip according to any one of claims 1-10.
12. The kit of claim 11, further comprising: diluting the 10 Xdiluent into 2 Xdiluent by using water without nuclease for dripping into a sample adding hole of the freeze-dried microchip, and then placing the freeze-dried microchip on a fluorescence PCR instrument for fluorescence PCR amplification.
13. The kit of claim 11 or 12, further comprising: mineral oil, sealing the sample application well on the lyophilized microchip;
positive control, specifically a mixture of classical strain of porcine reproductive and respiratory syndrome virus and genomic cDNA of NADC30-Like strain;
the negative control is in particular nuclease-free water.
14. Method for non-diagnostic purpose identification of classical strains of porcine reproductive and respiratory syndrome virus from NADC30-Like strains, characterized in that a lyophilized microchip according to any one of claims 1-10 and/or a kit according to any one of claims 11-13 is used for fluorescent PCR amplification of a sample to be tested.
15. The method of claim 14, wherein the fluorescent PCR amplification is performed by placing the lyophilized microchip on a fluorescent PCR instrument after the sample to be tested and the diluent are added to the sample addition well of the lyophilized microchip.
16. The method of claim 15, wherein the diluent is 10 x buffer.
17. The method of claim 15, wherein the fluorescent PCR amplification reaction procedure is: 50 ℃ for 10min;95 ℃ for 1min; the fluorescent signal was detected at the end of extension of each cycle by performing 40 cycles with 1 cycle at 95℃for 5s and 60℃for 15 s.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811052502.3A CN110885900B (en) | 2018-09-10 | 2018-09-10 | Freeze-dried microchip, kit and method for identifying classical strain of porcine reproductive and respiratory syndrome virus and NADC30-Like strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811052502.3A CN110885900B (en) | 2018-09-10 | 2018-09-10 | Freeze-dried microchip, kit and method for identifying classical strain of porcine reproductive and respiratory syndrome virus and NADC30-Like strain |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110885900A CN110885900A (en) | 2020-03-17 |
| CN110885900B true CN110885900B (en) | 2023-08-22 |
Family
ID=69745191
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811052502.3A Active CN110885900B (en) | 2018-09-10 | 2018-09-10 | Freeze-dried microchip, kit and method for identifying classical strain of porcine reproductive and respiratory syndrome virus and NADC30-Like strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110885900B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN114292961B (en) * | 2022-01-29 | 2024-08-13 | 龙岩学院 | Real-time fluorescent RT-PCR detection primer and probe set for identifying NADC-30-like strain |
| 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 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005035794A2 (en) * | 2003-10-09 | 2005-04-21 | Tetracore, Inc. | Detection of prrsv |
| CN101928782A (en) * | 2009-11-19 | 2010-12-29 | 金陵科技学院 | Kit for simultaneously detecting classical strains and variant strains of porcine reproductive and respiratory syndrome virus and detection method thereof |
| CN103205507A (en) * | 2013-03-08 | 2013-07-17 | 广西壮族自治区动物疫病预防控制中心 | RT-PCR primers and kit for detecting high-pathopoiesia porcine reproductive andrespiratory syndrome and classic porcine reproductive andrespiratory syndrome |
| CN104745726A (en) * | 2015-03-24 | 2015-07-01 | 北京佰鸥创投生物科技有限公司 | Triplet fluorescence quantitative detection kit for PRRSV (Porcine reproductive and respiratory syndrome virus), HP-PRRSV (Highly pathogenic porcine reproductive and respiratory syndrome virus) and CSFV (Classical Swine Fever Virus) |
| CN106868224A (en) * | 2017-04-20 | 2017-06-20 | 西南民族大学 | The RT PCR methods of detection pig blue-ear disease poison classical strainses, highly pathogenic mutant strain and the strains of NADC 30 |
-
2018
- 2018-09-10 CN CN201811052502.3A patent/CN110885900B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005035794A2 (en) * | 2003-10-09 | 2005-04-21 | Tetracore, Inc. | Detection of prrsv |
| CN101928782A (en) * | 2009-11-19 | 2010-12-29 | 金陵科技学院 | Kit for simultaneously detecting classical strains and variant strains of porcine reproductive and respiratory syndrome virus and detection method thereof |
| CN103205507A (en) * | 2013-03-08 | 2013-07-17 | 广西壮族自治区动物疫病预防控制中心 | RT-PCR primers and kit for detecting high-pathopoiesia porcine reproductive andrespiratory syndrome and classic porcine reproductive andrespiratory syndrome |
| CN104745726A (en) * | 2015-03-24 | 2015-07-01 | 北京佰鸥创投生物科技有限公司 | Triplet fluorescence quantitative detection kit for PRRSV (Porcine reproductive and respiratory syndrome virus), HP-PRRSV (Highly pathogenic porcine reproductive and respiratory syndrome virus) and CSFV (Classical Swine Fever Virus) |
| CN106868224A (en) * | 2017-04-20 | 2017-06-20 | 西南民族大学 | The RT PCR methods of detection pig blue-ear disease poison classical strainses, highly pathogenic mutant strain and the strains of NADC 30 |
Non-Patent Citations (1)
| Title |
|---|
| 微芯片实时荧光PCR 技术及其在动植物病害中的应用微芯片实时荧光PCR 技术及其在动植物病害中的应用;于水淼等;《中国化学会第九届全国仪器分析及样品预处理学术研讨会论文集》;20170624;摘要，引言部分第2段，第2.2节，第3节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110885900A (en) | 2020-03-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107475459B (en) | Detection method for simultaneously identifying American PRRSV classic strain, variant strain and novel virus type NADC30 strain | |
| CN110760620A (en) | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method | |
| US20230203575A1 (en) | Novel coronavirus rapid detection kit based on thermal convection pcr | |
| CN110885900B (en) | Freeze-dried microchip, kit and method for identifying classical strain of porcine reproductive and respiratory syndrome virus and NADC30-Like strain | |
| CN111334608B (en) | Dual-fluorescence freeze-drying microchip, kit and method for detecting novel coronavirus 2019-nCoV | |
| CN112795704B (en) | RAA primer pair, probe combination and kit for detecting porcine pseudorabies virus | |
| CN112176103A (en) | African swine fever virus fluorescence ERA constant temperature rapid detection kit | |
| CN110885904B (en) | Freeze-dried microchip, kit and method for identifying 16 pig disease pathogens | |
| CN112094953A (en) | Kit, primer and probe for simultaneously detecting bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus | |
| CN113249517A (en) | Primer, probe and kit for real-time fluorescent quantitative PCR (polymerase chain reaction) detection of bovine plague | |
| CN111321247B (en) | Freeze-drying microporous plate, kit and method for identifying African swine fever virus, swine fever wild strain and swine fever lapinized attenuated vaccine strain | |
| CN110885899B (en) | Freeze-drying microchip, kit and method for identifying 16 avian disease pathogens | |
| CN110885902B (en) | Freeze-drying microchip, kit and method for detecting porcine reproductive and respiratory syndrome virus and identifying highly pathogenic classical variant strain of porcine reproductive and respiratory syndrome virus | |
| CN115948575B (en) | Echinococcosis rrnS gene specific primer, probe combination, kit and application | |
| CN111926109B (en) | African swine fever virus fluorescence thermal convection PCR amplification primer pair, probe primer and prepared kit | |
| CN112063757B (en) | Primer and kit for detecting African swine fever virus and application of primer and kit | |
| CN114395643A (en) | Double-channel digital PCR detection kit and method for African swine fever virus | |
| CN109988853B (en) | Primer and probe combination for chlamydia psittaci genotype detection and application | |
| CN112522446A (en) | Detection primer pair and kit for wild strain of porcine pseudorabies virus | |
| CN113388700A (en) | Kit for detecting FCV (FCV), FPV (FPV) and FHV-1 viruses by using nucleic acid hand-free triple fluorescence RT-LAMP (reverse transcription loop-mediated isothermal amplification) | |
| CN111500773A (en) | Fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) primer, probe and kit for identifying serotype of epidemic hemorrhagic disease virus | |
| CN116083652B (en) | Primer, fluorescent freeze-dried microchip, kit and method for detecting monkey pox virus | |
| CN106701966B (en) | Rapid detection method for pathogenic microorganisms based on analysis of PCR (polymerase chain reaction) byproduct pyrophosphoric acid | |
| CN111996293A (en) | Primer and kit for efficiently detecting porcine epidemic encephalitis B virus | |
| CN110885903B (en) | Freeze-drying microchip, kit and method for identifying H9 and H6 subtype low-pathogenicity avian influenza virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |