CN110885904B - Freeze-dried microchip, kit and method for identifying 16 pig disease pathogens - Google Patents

Freeze-dried microchip, kit and method for identifying 16 pig disease pathogens Download PDF

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CN110885904B
CN110885904B CN201811053108.1A CN201811053108A CN110885904B CN 110885904 B CN110885904 B CN 110885904B CN 201811053108 A CN201811053108 A CN 201811053108A CN 110885904 B CN110885904 B CN 110885904B
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microchip
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swine
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CN110885904A (en
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王新杰
高姗姗
孙晓明
胡祥钰
韩焘
周智
倪建强
刘玉良
王传彬
杨林
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China Animal Disease Control And Prevention Center (agricultural Slaughter Technology Center)
Beijing Yisen Biotechnology Co ltd
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Beijing Yisen Biotechnology Co ltd
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Abstract

The invention discloses a freeze-dried microchip, a kit and a method for identifying 16 pig disease pathogens, wherein the kit comprises a freeze-dried microchip, a bottle of mineral oil, a tube of diluent and a tube of nuclease-free water, and the freeze-dried microchip is coated with a primer, a probe, taq enzyme, reverse transcriptase, trehalose and Tris-Cl, dNTP, mg 2+ . The lyophilization conditions were: 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 analysis and drying stage, the temperature of the partition board is raised to-25 ℃ for 1h, then the temperature of the partition board is raised to 37 ℃ for 2h, finally the partition board is lowered to 25 ℃ for 1h, and the freeze-dried microchip is obtained, and the diluent and the sample nucleic acid are added when the micro-chip is used. The detection kit can detect 16 pig disease pathogens simultaneously, and has high accuracy, specificity and sensitivity and short detection time.

Description

Freeze-dried microchip, kit and method for identifying 16 pig disease pathogens
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 16 swine pathogens.
Background
The pig breeding is mainly large-scale intensive breeding in China, and the high-density breeding mode also makes the infectious diseases of pigs difficult to prevent and the problems are increasingly remarkable. And multiple pathogens are infected simultaneously, so that detection and prevention of diseases are difficult.
Common infectious disease pathogens in pigs are porcine Foot and mouth disease virus (Foot-and-mouth disease virus, FMDV), porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), porcine transmissible gastroenteritis virus (Transmissible gastroenteritis virus, TEGV), porcine coronavirus (Porcine coronavirus, DCV), porcine encephalitis virus (Japanese encephalitis virus, JEV), mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, MHP), porcine rotavirus (Porcine rotavirus, PRTV), swine fever virus (Classical swine fever virus, CSFV), swine influenza virus (Swine Influenza virus, SIV), porcine parvovirus (Porcine parvovirus, PPV), porcine circovirus (Porcine circovirus, PCV), porcine pseudorabies virus (Porcine pseudorabies virus, PRV), porcine actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae, APP), porcine streptococcus (Streptococcus suis, SS), haemophilus parasuis (Haemophilus parasuis, HPS), and chlamydia (Swine Chlamydiosis, SC). In recent years, although various nations scientists have conducted extensive studies on swine diseases and various pathogens. At present, the method adopted for detecting the swine pathogens in China establishes serological detection technologies such as separation, purification, culture and identification of the pathogens, an indirect enzyme-linked immunosorbent assay (ELISA) and the like and a molecular detection technology (RT-PCR) in recent years. 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 used as a novel molecular biology technology, since the birth, a large number of target fragments can be obtained through gene amplification in a short time due to high specificity and sensitivity, the defect that the traditional pig pathogen detection technology comprises long virus separation and identification test period is overcome, and a sensitive, rapid and practical detection method is provided for the early detection of pig pathogens, so that the method becomes one of the important means for detecting pig pathogens at present. However, the common PCR still has the defects of complicated operation, long detection time, easy pollution and the like. So the integrated reagent products which are fast, accurate, high in sensitivity, portable and capable of detecting and identifying the 16 swine diseases simultaneously in field operation are urgently needed in the market at present. The method can provide advanced and effective early rapid detection technology and monitoring means for prevention and control of swine diseases in China, and meanwhile, can also strive for valuable rapid reaction time for prevention and control of swine diseases.
Disclosure of Invention
Based on the above-mentioned needs in the art, the present invention aims to provide a lyophilized microchip and a kit for identifying 16 swine pathogens, so that the lyophilized microchip and the kit can rapidly and effectively detect swine pathogens, and have high accuracy, specificity and sensitivity, and good repeatability.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a lyophilized microchip for identifying 16 swine pathogens, wherein a fluorescent PCR reaction system for identifying 16 swine pathogens is immobilized on the microchip by lyophilization;
the fluorescent PCR reaction liquid comprises the following components: the following primers and probes:
FMDV-F:5’-GGCCGAGACCACAAATGTG-3’；
FMDV-R:5’-TCGGCCTTGCCATGTGTAA-3’；
FMDV-Taqman:5’-AGGGATGGGTCTGCTT-3’；
PEDV-F:5’-GCAAATACATTGGCAGCATAACC-3’；
PEDV-R:5’-TGCTGGTGAGGATGGTCTTTC-3’；
PEDV-Taqman:5’-ATGCAATTAGCTGTACAGTGT-3’；
TEGV-F:5’-TGGTGAGCCCTTGCAAATG-3’；
TEGV-R:5’-CAGTTACAACAACCCGCAATTTT-3’；
TEGV-Taqman:5’-TAATAGCACCTTCAGCAGAAT-3’；
DCV-F:5’-AGCCACCCACCAAACCAA-3’；
DCV-R:5’-TGGGTTTAGCAGACTGGTCTTGT-3’；
DCV-Taqman:5’-AGGACAAGAAGCCTGAC-3’；
JEV-F:5’-CGCGGACCAGGCATTC-3’；
JEV-R:5’-GAACTCTCCCCATGTGTTTGGA-3’；
JEV-Taqman:5’-AAGCGAAGCAGGAGAT-3’；
MHP-F:5’-TGACCGTCTTAACTCGCCAAAT-3’；
MHP-R:5’-GGCGGTAGGGAATTAAAACTATTG-3’；
MHP-Taqman:5’-CGCAAGTCTTGATAAAA-3’；
PRTV-F:5’-TTTGTTTACATTGACTACTGGGATGAT-3’；
PRTV-R:5’-AGCAGCAAGTGACCGAACATATAC-3’；
PRTV-Taqman:5’-ACAAGCATTCAGAAACA-3’；
CSFV-F：5’-TTTGCGTGGCAGGTTCCT-3’；
CSFV-R：5’-AATGATGCCAAATACCTCCTACTGA-3’；
CSFV-Taqman:5’-AAAGTCACAGCACTTAAT-3’；
SIV-F:5’-CCGAAATCGCGCAGAGA-3’；
SIV-R:5’-CATGAGAGCCTCAAGATCTGTGTT-3’；
SIV-Taqman:5’-TGAAGATGTCTTTGCTGGAA-3’；
PPV-F:5’-GCAGCTAACACAAGAAAAGGTTATCAC-3’；
PPV-R:5’-CCTGAGCTGGCCTAATTGCT-3’；
PPV-Taqman:5’-TAATAGCTACACAGAAGCAA-3’；
PCV-F:5’-TGAGCGGGAAAATGCAGAA-3’；
PCV-R:5’-CCAGGTGGCCCCACAA-3’；
PCV-Taqman:5’-TGGAAGACGAATGTACACG-3’；
PRV-F:5’-CGTCGTGAGCAGCATGAT-3’；
PRV-R:5’-CCATGATGACCAGCACGAT-3’；
PRV-Taqman:5’-CATCGGGATCCTGGC-3’；
APP-F:5’-GGTACCGGTCTTAGGTATCATTGAA-3’；
APP-R:5’-CTTCGTGGTGACCGCAATTT-3’；
APP-Taqman:5’-CGTGCATATCTGCC-3’；
SS-F:5’-TTCGAAGCAACGCGAAGAA-3’；
SS-R:5’-CCCATCTCTAGGGCGGTCAT-3’；
SS-Taqman:5’-CCAGGTCTTGACATCC-3’；
HPS-F:5’-GCGGCAGGCTTAACACATG-3’；
HPS-R:5’-CGTCAGCAAAGAAAGCAAGCT-3’；
HPS-Taqman:5’-TCGAACGGTAGCAGGGA-3’；
SC-F:5’-CACAGTCCGTTGTCGAGCTTTA-3’；
SC-R:5’-GCGGCTCTAGCACCAACAC-3’；
SC-Taqman:5’-CAGATACTGCCTTTGCTT-3’。
the fluorescent PCR reaction system further comprises: taq enzyme, reverse transcriptase, trehalose, tris-Cl, dNTP, mg 2+ ；
Preferentially, the 5' -end of the Taqman probe of the porcine foot-and-mouth disease virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine coronavirus, porcine Japanese encephalitis virus, porcine mycoplasma pneumoniae, porcine rotavirus, swine fever virus, porcine influenza virus, porcine parvovirus, porcine circovirus, porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae, porcine streptococcus, haemophilus parasuis and porcine chlamydia is marked with a fluorescence report group; 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-Cl 5mM, template 5 ng/. Mu.L balance sterilized deionized water;
preferably, the template is selected from cDNA of 16 pig disease pathogens, cDNA of a sample to be detected and sterilized deionized water, and is respectively used as positive control, sample detection system and negative control;
more preferably, the upstream primer, the downstream primer and the Taqman probe of the fluorescent PCR reaction system in each sample-adding well before the microchip is freeze-dried respectively comprise a primer group and a Taqman probe of 2 swine pathogens in 16 swine pathogens, and the templates of the fluorescent PCR reaction system of the positive control are respectively cDNA mixtures of 2 swine pathogens in 16 swine pathogens; of the 16 swine pathogens, the cDNA mixture of the primer pairs and Taqman probes for the 2 swine pathogens in each of the following groups and templates for positive controls were placed in the same well on the microchip: foot and mouth disease virus and porcine epidemic diarrhea virus; transmissible gastroenteritis virus and porcine coronavirus; encephalitis b virus and mycoplasma hyopneumoniae; porcine rotavirus and swine fever virus; swine influenza virus and swine parvovirus; porcine circovirus and porcine pseudorabies virus; actinobacillus pleuropneumoniae and streptococcus suis; haemophilus parasuis and chlamydia suis.
Preferentially, the fluorescent reporter group marked at the 5' end of the Taqman probes of the foot-and-mouth disease virus, transmissible gastroenteritis virus, porcine Japanese encephalitis virus, porcine rotavirus, porcine influenza virus, porcine circovirus, porcine actinobacillus pleuropneumoniae and haemophilus parasuis is a FAM fluorescent reporter group;
the fluorescent reporter group marked at the 5' end of the Taqman probes of the porcine epidemic diarrhea virus, porcine coronavirus, mycoplasma hyopneumoniae, swine fever virus, porcine parvovirus, porcine pseudorabies virus, streptococcus suis and chlamydia suis is a ROX fluorescent reporter group;
the Taqman probe 3' -end-labeled fluorescence quenching group of the porcine foot-and-mouth disease virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine coronavirus, porcine Japanese encephalitis virus, porcine mycoplasma pneumoniae, porcine rotavirus, swine fever virus, porcine influenza virus, porcine parvovirus, porcine circovirus, porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae, porcine streptococcus, haemophilus parasuis and porcine chlamydia is an MGB quenching group.
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 24; 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.
A kit for identifying 16 swine pathogens comprising the lyophilized 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;
the method for identifying 16 pig disease pathogens is characterized in that the freeze-dried microchip and/or the kit is used for carrying 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 PCR detection kit of 16 swine disease pathogens fluorescent freeze-dried microchips, which comprises a PCR freeze-dried microchip, wherein the PCR freeze-dried microchip comprises primers with the following nucleotide sequences and Taqman probes:
FMDV-F:5’-GGCCGAGACCACAAATGTG-3’；
FMDV-R:5’-TCGGCCTTGCCATGTGTAA-3’；
FMDV-Taqman:5’-AGGGATGGGTCTGCTT-3’；
PEDV-F:5’-GCAAATACATTGGCAGCATAACC-3’；
PEDV-R:5’-TGCTGGTGAGGATGGTCTTTC-3’；
PEDV-Taqman:5’-ATGCAATTAGCTGTACAGTGT-3’；
TEGV-F:5’-TGGTGAGCCCTTGCAAATG-3’；
TEGV-R:5’-CAGTTACAACAACCCGCAATTTT-3’；
TEGV-Taqman:5’-TAATAGCACCTTCAGCAGAAT-3’；
DCV-F:5’-AGCCACCCACCAAACCAA-3’；
DCV-R:5’-TGGGTTTAGCAGACTGGTCTTGT-3’；
DCV-Taqman:5’-AGGACAAGAAGCCTGAC-3’；
JEV-F:5’-CGCGGACCAGGCATTC-3’；
JEV-R:5’-GAACTCTCCCCATGTGTTTGGA-3’；
JEV-Taqman:5’-AAGCGAAGCAGGAGAT-3’；
MHP-F:5’-TGACCGTCTTAACTCGCCAAAT-3’；
MHP-R:5’-GGCGGTAGGGAATTAAAACTATTG-3’；
MHP-Taqman:5’-CGCAAGTCTTGATAAAA-3’；
PRTV-F:5’-TTTGTTTACATTGACTACTGGGATGAT-3’；
PRTV-R:5’-AGCAGCAAGTGACCGAACATATAC-3’；
PRTV-Taqman:5’-ACAAGCATTCAGAAACA-3’；
CSFV-F：5’-TTTGCGTGGCAGGTTCCT-3’；
CSFV-R：5’-AATGATGCCAAATACCTCCTACTGA-3’；
CSFV-Taqman:5’-AAAGTCACAGCACTTAAT-3’；
SIV-F:5’-CCGAAATCGCGCAGAGA-3’；
SIV-R:5’-CATGAGAGCCTCAAGATCTGTGTT-3’；
SIV-Taqman:5’-TGAAGATGTCTTTGCTGGAA-3’；
PPV-F:5’-GCAGCTAACACAAGAAAAGGTTATCAC-3’；
PPV-R:5’-CCTGAGCTGGCCTAATTGCT-3’；
PPV-Taqman:5’-TAATAGCTACACAGAAGCAA-3’；
PCV-F:5’-TGAGCGGGAAAATGCAGAA-3’；
PCV-R:5’-CCAGGTGGCCCCACAA-3’；
PCV-Taqman:5’-TGGAAGACGAATGTACACG-3’；
PRV-F:5’-CGTCGTGAGCAGCATGAT-3’；
PRV-R:5’-CCATGATGACCAGCACGAT-3’；
PRV-Taqman:5’-CATCGGGATCCTGGC-3’；
APP-F:5’-GGTACCGGTCTTAGGTATCATTGAA-3’；
APP-R:5’-CTTCGTGGTGACCGCAATTT-3’；
APP-Taqman:5’-CGTGCATATCTGCC-3’；
SS-F:5’-TTCGAAGCAACGCGAAGAA-3’；
SS-R:5’-CCCATCTCTAGGGCGGTCAT-3’；
SS-Taqman:5’-CCAGGTCTTGACATCC-3’；
HPS-F:5’-GCGGCAGGCTTAACACATG-3’；
HPS-R:5’-CGTCAGCAAAGAAAGCAAGCT-3’；
HPS-Taqman:5’-TCGAACGGTAGCAGGGA-3’；
SC-F:5’-CACAGTCCGTTGTCGAGCTTTA-3’；
SC-R:5’-GCGGCTCTAGCACCAACAC-3’；
SC-Taqman:5’-CAGATACTGCCTTTGCTT-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 fluorescent reporter group marked at the 5' end of the Taqman probes of the foot-and-mouth disease virus, transmissible gastroenteritis virus, porcine Japanese encephalitis virus, porcine rotavirus, porcine influenza virus, porcine circovirus, porcine actinobacillus pleuropneumoniae and haemophilus parasuis is a FAM fluorescent reporter group; the 5' end of the nucleotide sequence of the Taqman probe for detecting porcine epidemic diarrhea virus, porcine coronavirus, mycoplasma hyopneumoniae, swine fever virus, porcine parvovirus, porcine pseudorabies virus, streptococcus suis and chlamydia is marked with a ROX fluorescence report group.
The freeze-dried microchip reagent also comprises DNA polymerase, reverse transcriptase, dNTP and Mg 2+ Trehalose, tris-Cl.
The whole freeze-dried microchip sample loading holes of the AriaYSB freeze-dried microchip of the fluorescence quantitative PCR are 24, the fluorescence PCR reaction system is divided into 8 groups, and the fluorescence PCR reaction system respectively comprises 2 primer probes of porcine foot-and-mouth disease virus and porcine epidemic diarrhea virus, 2 primer probes of transmissible gastroenteritis virus and porcine coronavirus, 2 primer probes of porcine Japanese encephalitis virus and mycoplasma hyopneumoniae, 2 primer probes of porcine rotavirus and swine fever virus, 2 primer probes of swine influenza virus and porcine parvovirus, 2 primer probes of porcine circovirus and porcine pseudorabies virus, 2 primer probes of porcine actinobacillus pleuropneumoniae and swine streptococcus, and 2 primer probes of haemophilus parasuis and swine chlamydia.
The freeze-dried microchip is divided into a positive control well, a sample well and a negative control well. Wherein the positive control well further comprises sample DNA/cDNA.
The positive control is a foot-and-mouth disease virus and porcine epidemic diarrhea virus genome cDNA mixture; a mixture of porcine transmissible gastroenteritis virus genomic cDNA and porcine coronavirus genomic DNA; a mixture of swine Japanese encephalitis virus genomic cDNA and swine mycoplasma pneumoniae genomic DNA; porcine rotavirus and swine fever virus genomic cDNA mixtures; swine influenza virus genomic cDNA and swine parvovirus genomic DNA mixture; porcine circovirus and porcine pseudorabies virus genomic DNA mixtures; a mixture of actinobacillus pleuropneumoniae and streptococcus suis genomic DNA; haemophilus parasuis and chlamydia suis genomic DNA mixtures.
The total volume of each microchip well was 2. Mu.L.
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 conventional reagents comprise commercial molecular reagents including mineral oil, diluent and water without nuclease;
the commercial dilutions were 10 Xbuffer, diluted with nuclease-free water to 2 Xbuffer and 1 Xbuffer.
The 2 mu L reaction system of the freeze-dried microchip fluorescence quantitative PCR comprises: freeze-drying positive control holes and negative control holes by fluorescent quantitative PCR, wherein 2 mu L of 1 Xbuffer diluent; the sample wells were lyophilized by fluorescent quantitative PCR, 2 Xbuffer dilution 1. Mu.L, and sample DNA/RNA 1. Mu.L was detected.
The 16 swine pathogen freeze-dried microchip fluorescent RT-PCR detection kit is characterized in that the primer and/or the kit are/is adopted to detect a sample to be detected.
The detection refers to the fluorescent quantitative PCR detection of the freeze-dried microchip; the fluorescent PCR reaction system adopted before freeze-drying comprises the following components: positive control wells 2 upstream primers each 0.4 μm;2 downstream primers each 0.4. Mu.M; 2 Taqman probes 0.4. Mu.M each; 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, positive control 5 ng/. Mu.L; the balance of sterilized deionized water, wherein the volume of each hole is 2 mu L; sample wells and negative control wells contained 0.4 μm each of 2 upstream primers; 2 downstream primers each 0.4. Mu.M; 2 Taqman probes each 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, wherein the volume of each hole is 2 mu L;
the fluorescent RT-PCR reaction conditions 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.
By adopting the technical scheme, the invention has at least the following advantages:
(1) According to the domestic discovered pig foot-and-mouth disease virus, pig epidemic diarrhea virus, transmissible gastroenteritis virus, pig coronavirus, pig Japanese encephalitis virus, pig mycoplasma pneumoniae, pig rotavirus, swine fever virus, pig influenza virus, pig parvovirus, pig circovirus, pig pseudorabies virus, pig actinobacillus pleuropneumoniae, pig streptococcus, haemophilus parasuis and pig chlamydia pathogen conserved genes, primers are designed, specific primers and Taqman-MGB probes are synthesized, and 16 pig disease pathogens can be detected rapidly and sensitively simultaneously by adopting a fluorescent quantitative PCR method, and the method has high accuracy, specificity and sensitivity and good 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, only 2 mu L and 20-25 mu L of conventional reaction system, so that the microchip technology can save the use amount of RT-PCR amplification reagents and 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.
(7) The positive control and the negative control are freeze-dried in the microchip together with the reaction reagent, and only diluent and a sample to be detected are needed to be added when the sample is detected, and the negative control and the positive control are not needed to be added. The program is a fixed setting program, the program can be detected by calling the existing program of the software without setting, and the detection time is further shortened.
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 reagent, positive control and negative control are freeze-dried on the microchip, and only sample holes are needed to be added during sample loading. In combination with Taqman probe fluorescent quantitative PCR technology, the analysis of nucleic acids is that the microchip simultaneously reads the fluorescent signal generated by the PCR products on each reactor during thermal cycling. 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 16 pig disease pathogens 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 invention develops a microchip fluorescence quantitative PCR reaction system capable of rapidly and effectively detecting 16 swine pathogens 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 10TCID 50 And the sensitivity is very high, the variation coefficient between batches of the detection kit is between 0.25 and 1.95 percent, and the repeatability is good.
Drawings
FIG. 1 shows the specific detection of 16 swine disease pathogen freeze-dried microchips for swine foot and mouth disease virus. In the figure, 1: positive control of swine foot-and-mouth disease virus; 2: porcine foot-and-mouth disease virus sample nucleic acid; 3: pig foot and mouth disease virus negative control.
FIG. 2 is a specific assay of 16 swine disease pathogen lyophilized microchips for swine foot and mouth disease virus. In the figure, 1: positive control of swine foot-and-mouth disease virus; 2: porcine epidemic diarrhea virus sample nucleic acid; 3: pig foot and mouth disease virus negative control.
FIG. 3 is a specific assay of 16 swine disease pathogen lyophilized microchips for swine foot and mouth disease virus. In the figure, 1: positive control of swine foot-and-mouth disease virus; 2: porcine transmissible gastroenteritis virus; 3: porcine coronavirus; 4: porcine Japanese encephalitis virus; 5: mycoplasma hyopneumoniae; 6: porcine rotavirus; 7: swine fever virus; 8: swine influenza virus; 9: porcine parvovirus; 10: porcine circovirus; 11: porcine pseudorabies virus; 12: actinobacillus pleuropneumoniae; 13: streptococcus suis; 14: haemophilus parasuis; 15: chlamydia suis; 16: porcine reproductive and respiratory syndrome virus; 17: pig foot and mouth disease virus negative control.
FIG. 4 is a specific assay of 16 porcine pathogen freeze-dried microchips for porcine epidemic diarrhea virus. In the figure, 1: positive control of porcine epidemic diarrhea virus; 2: porcine epidemic diarrhea virus sample nucleic acid; 3: porcine epidemic diarrhea virus negative control.
FIG. 5 is a specific assay of 16 porcine pathogen freeze-dried microchips for porcine epidemic diarrhea virus. In the figure, 1: positive control of porcine epidemic diarrhea virus; 2: porcine foot-and-mouth disease virus sample nucleic acid; 3: porcine epidemic diarrhea virus negative control.
FIG. 6 is a specific assay of 16 swine disease pathogen lyophilized microchips for swine foot and mouth disease virus. In the figure, 1: positive control of porcine epidemic diarrhea virus; 2: porcine transmissible gastroenteritis virus; 3: porcine coronavirus; 4: porcine Japanese encephalitis virus; 5: mycoplasma hyopneumoniae; 6: porcine rotavirus; 7: swine fever virus; 8: swine influenza virus; 9: porcine parvovirus; 10: porcine circovirus; 11: porcine pseudorabies virus; 12: actinobacillus pleuropneumoniae; 13: streptococcus suis; 14: haemophilus parasuis; 15: chlamydia suis; 16: porcine reproductive and respiratory syndrome virus; 17: porcine epidemic diarrhea virus negative control.
FIG. 7 shows the result of detecting sensitivity of the swine foot-and-mouth disease virus by using the lyophilized 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: nuclease-free water.
FIG. 8 shows the detection of susceptibility of porcine foot-and-mouth disease virus by conventional fluorescent RT-PCR reagentSexual results. 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°TCID 50 /mL; h: nuclease-free water.
FIG. 9 shows the standard curve results of the detection of the porcine foot-and-mouth disease virus by using the lyophilized microchip fluorescent RT-PCR reagent.
FIG. 10 shows the standard curve results of the detection of the porcine foot-and-mouth disease 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 epidemic diarrhea virus, porcine rotavirus, swine fever virus, porcine parvovirus, porcine circovirus and porcine pseudorabies virus inactivated strain used in the experimental example of the invention are preserved by the national animal epidemic, porcine foot and mouth disease virus, transmissible gastroenteritis virus, porcine coronavirus, porcine Japanese encephalitis virus and porcine influenza virus inactivated strain, porcine mycoplasma pneumoniae, porcine chlamydia inactivated pathogen and porcine actinobacillus pleuropneumoniae, porcine streptococcus and haemophilus parasuis inactivated bacterial strain are preserved by Beijing Yingsen biotechnology Co Ltd, and all strains, mycoplasma and chlamydia used in the invention are commercially available.
Group 1 examples: the freeze-dried microchip of the present invention
The present group of examples provides a lyophilized microchip for identifying 16 swine pathogens, characterized in that a fluorescent PCR reaction system for identifying 16 swine pathogens is immobilized on the microchip by lyophilization;
the fluorescent PCR reaction liquid comprises the following components: the following primers and probes: FMDV-F5'-GGCCGAGACCACAAATGTG-3';
FMDV-R:5’-TCGGCCTTGCCATGTGTAA-3’；
FMDV-Taqman:5’-AGGGATGGGTCTGCTT-3’；
PEDV-F:5’-GCAAATACATTGGCAGCATAACC-3’；
PEDV-R:5’-TGCTGGTGAGGATGGTCTTTC-3’；
PEDV-Taqman:5’-ATGCAATTAGCTGTACAGTGT-3’；
TEGV-F:5’-TGGTGAGCCCTTGCAAATG-3’；
TEGV-R:5’-CAGTTACAACAACCCGCAATTTT-3’；
TEGV-Taqman:5’-TAATAGCACCTTCAGCAGAAT-3’；
DCV-F:5’-AGCCACCCACCAAACCAA-3’；
DCV-R:5’-TGGGTTTAGCAGACTGGTCTTGT-3’；
DCV-Taqman:5’-AGGACAAGAAGCCTGAC-3’；
JEV-F:5’-CGCGGACCAGGCATTC-3’；
JEV-R:5’-GAACTCTCCCCATGTGTTTGGA-3’；
JEV-Taqman:5’-AAGCGAAGCAGGAGAT-3’；
MHP-F:5’-TGACCGTCTTAACTCGCCAAAT-3’；
MHP-R:5’-GGCGGTAGGGAATTAAAACTATTG-3’；
MHP-Taqman:5’-CGCAAGTCTTGATAAAA-3’；
PRTV-F:5’-TTTGTTTACATTGACTACTGGGATGAT-3’；
PRTV-R:5’-AGCAGCAAGTGACCGAACATATAC-3’；
PRTV-Taqman:5’-ACAAGCATTCAGAAACA-3’；
CSFV-F：5’-TTTGCGTGGCAGGTTCCT-3’；
CSFV-R：5’-AATGATGCCAAATACCTCCTACTGA-3’；
CSFV-Taqman:5’-AAAGTCACAGCACTTAAT-3’；
SIV-F:5’-CCGAAATCGCGCAGAGA-3’；
SIV-R:5’-CATGAGAGCCTCAAGATCTGTGTT-3’；
SIV-Taqman:5’-TGAAGATGTCTTTGCTGGAA-3’；
PPV-F:5’-GCAGCTAACACAAGAAAAGGTTATCAC-3’；
PPV-R:5’-CCTGAGCTGGCCTAATTGCT-3’；
PPV-Taqman:5’-TAATAGCTACACAGAAGCAA-3’；
PCV-F:5’-TGAGCGGGAAAATGCAGAA-3’；
PCV-R:5’-CCAGGTGGCCCCACAA-3’；
PCV-Taqman:5’-TGGAAGACGAATGTACACG-3’；
PRV-F:5’-CGTCGTGAGCAGCATGAT-3’；
PRV-R:5’-CCATGATGACCAGCACGAT-3’；
PRV-Taqman:5’-CATCGGGATCCTGGC-3’；
APP-F:5’-GGTACCGGTCTTAGGTATCATTGAA-3’；
APP-R:5’-CTTCGTGGTGACCGCAATTT-3’；
APP-Taqman:5’-CGTGCATATCTGCC-3’；
SS-F:5’-TTCGAAGCAACGCGAAGAA-3’；
SS-R:5’-CCCATCTCTAGGGCGGTCAT-3’；
SS-Taqman:5’-CCAGGTCTTGACATCC-3’；
HPS-F:5’-GCGGCAGGCTTAACACATG-3’；
HPS-R:5’-CGTCAGCAAAGAAAGCAAGCT-3’；
HPS-Taqman:5’-TCGAACGGTAGCAGGGA-3’；
SC-F:5’-CACAGTCCGTTGTCGAGCTTTA-3’；
SC-R:5’-GCGGCTCTAGCACCAACAC-3’；
SC-Taqman:5’-CAGATACTGCCTTTGCTT-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 the Taqman probes of the 16 swine pathogens is marked with a fluorescence report group; the 3' -end is marked with a fluorescence quenching group.
In a more specific embodiment, the fluorescent PCR reaction systems are divided into 8 groups, respectively: foot-and-mouth disease virus of pig and pigEpidemic diarrhea virus, transmissible gastroenteritis virus and porcine coronavirus, porcine Japanese encephalitis virus and mycoplasma hyopneumoniae, porcine rotavirus and swine fever virus, porcine influenza virus and porcine parvovirus, porcine circovirus and porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae and streptococcus suis, haemophilus parasuis and chlamydia suis. Each set of fluorescent PCR reaction systems included: positive control fluorescent PCR reaction system, sample reaction system and negative control reaction system. The positive control fluorescent PCR reaction system comprises 0.4 mu M of upstream primers (2 types); downstream primer (2) 0.4. Mu.M; taqman probe 0.4. Mu.M (2); 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, positive control 5 ng/. Mu.L, balance sterile deionized water, total volume 2. Mu.L; the sample reaction system and the negative control reaction system contained 0.4. Mu.M of the upstream primer (2 kinds); downstream primer (2) 0.4. Mu.M; taqman probe 0.4. Mu.M (2); 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, and the total volume is 2 mu L;
the positive control is a foot-and-mouth disease virus and porcine epidemic diarrhea virus genome cDNA mixture respectively; a mixture of porcine transmissible gastroenteritis virus genomic cDNA and porcine coronavirus genomic DNA; a mixture of swine Japanese encephalitis virus genomic cDNA and swine mycoplasma pneumoniae genomic DNA; porcine rotavirus and swine fever virus genomic cDNA mixtures; swine influenza virus genomic cDNA and swine parvovirus genomic DNA mixture; porcine circovirus and porcine pseudorabies virus genomic DNA mixtures; a mixture of actinobacillus pleuropneumoniae and streptococcus suis genomic DNA; haemophilus parasuis and chlamydia suis genomic DNA mixtures.
The final concentration of the components is not only the final concentration of a fluorescent PCR reaction system before freeze-drying, but also the final concentration of the components after freeze-drying, and 1 Xbuffer 2 mu L is added to each of positive control holes and negative control holes during sample loading; sample wells 2. Mu.L of each well was added to the sample wells after mixing 2 Xbuffer and nucleic acid extract at 1:1.
Preferentially, the fluorescent reporter group marked at the 5' end of the Taqman probes of the foot-and-mouth disease virus, transmissible gastroenteritis virus, porcine Japanese encephalitis virus, porcine rotavirus, porcine influenza virus, porcine circovirus, porcine actinobacillus pleuropneumoniae and haemophilus parasuis is a FAM fluorescent reporter group;
the fluorescent reporter group marked at the 5' end of the Taqman probes of the porcine epidemic diarrhea virus, porcine coronavirus, mycoplasma hyopneumoniae, swine fever virus, porcine parvovirus, porcine pseudorabies virus, streptococcus suis and chlamydia suis is a ROX fluorescent reporter group;
the Taqman probe 3' -end-labeled fluorescence quenching group of the porcine foot-and-mouth disease virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine coronavirus, porcine Japanese encephalitis virus, porcine mycoplasma pneumoniae, porcine rotavirus, swine fever virus, porcine influenza virus, porcine parvovirus, porcine circovirus, porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae, porcine streptococcus, haemophilus parasuis and porcine chlamydia is an MGB quenching group.
In some embodiments, the microchip is provided with a plurality of sample application holes; 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 present invention, and use them for qualitative or quantitative detection of 16 swine pathogens, so that the expected effect according to the present invention can be obtained, and thus any commercially synthesized primers are put into a commercial package labeled with the purpose of "detecting 16 swine pathogens", or the use of primers using the above sequences for commercial detection of 16 swine pathogens falls within the scope of the claimed invention.
Group 2 examples: the kit of the invention
This set of examples provides a kit for identifying 16 swine pathogens comprising a lyophilized microchip according to any one of the examples of set 1.
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. Diluting with water without nuclease, dripping into sample-adding hole of lyophilized microchip, and placing the lyophilized microchip on fluorescent PCR instrument for fluorescent PCR amplification.
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.
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, dabcyl 2; 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, diluents, double distilled water;
in a further preferred embodiment, the commercial diluent is 10 Xbuffer (without Mg 2+ ) The solution was diluted with nuclease-free water to 2 Xbuffer and 1 Xbuffer. 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 present set of examples provides a method for fluorescent RT-PCR detection of lyophilized microchips for the identification of highly pathogenic 16 swine pathogens. 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 dilution 1 Xbuffer positive control and negative control wells are added in an amount of 2. Mu.L. Mixing the diluted solution 2 Xbuffer with the nucleic acid extract at a ratio of 1:1, and adding 2 μl;
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 16 pig disease pathogens comprises: upstream primer 0.4. Mu.M; 0.4. Mu.M of downstream primer; t (T)aqman probe 0.4 μm; 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; if the positive control hole has 5 ng/. Mu.L of corresponding pathogenic DNA or cDNA; the balance was sterilized deionized water, and the total volume was 2. Mu.L.
The whole freeze-dried microchip sample loading holes of the freeze-dried microchip of the fluorescent quantitative PCR are 24, the fluorescent PCR reaction system is divided into 8 groups, and the kit respectively comprises 2 primer probes of porcine foot-and-mouth disease virus and porcine epidemic diarrhea virus, 2 primer probes of porcine transmissible gastroenteritis virus and porcine coronavirus, 2 primer probes of porcine Japanese encephalitis virus and porcine mycoplasma pneumoniae, 2 primer probes of porcine rotavirus and swine fever virus, 2 primer probes of porcine influenza virus and porcine parvovirus, 2 primer probes of porcine circovirus and porcine pseudorabies virus, 2 primer probes of porcine actinobacillus pleuropneumoniae and porcine streptococcus, and 2 primer probes of haemophilus parasuis and porcine chlamydia.
The freeze-dried microchip is divided into a positive control well, a sample well and a negative control well. Wherein the positive control well further comprises sample DNA/cDNA.
The total volume of each microchip well was 2. Mu.L.
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 specificity verification of 16 swine pathogen freeze-dried microchip fluorescent RT-PCR detection kit
1. Design of primers and Taqman-MGB probes
A plurality of pairs of primers and probes are designed according to the domestic discovered pathogenic conserved genes of porcine foot-and-mouth disease virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine coronavirus, porcine Japanese encephalitis virus, mycoplasma hyopneumoniae, porcine rotavirus, swine fever virus, swine influenza virus, porcine parvovirus, porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae, porcine streptococcus, haemophilus parasuis and porcine chlamydia. And finally, respectively determining a group of optimal primers and a Taqman-MGB probe through comparison and screening.
FMDV-F:5’-GGCCGAGACCACAAATGTG-3’；
FMDV-R:5’-TCGGCCTTGCCATGTGTAA-3’；
FMDV-Taqman:5’-AGGGATGGGTCTGCTT-3’；
PEDV-F:5’-GCAAATACATTGGCAGCATAACC-3’；
PEDV-R:5’-TGCTGGTGAGGATGGTCTTTC-3’；
PEDV-Taqman:5’-ATGCAATTAGCTGTACAGTGT-3’；
TEGV-F:5’-TGGTGAGCCCTTGCAAATG-3’；
TEGV-R:5’-CAGTTACAACAACCCGCAATTTT-3’；
TEGV-Taqman:5’-TAATAGCACCTTCAGCAGAAT-3’；
DCV-F:5’-AGCCACCCACCAAACCAA-3’；
DCV-R:5’-TGGGTTTAGCAGACTGGTCTTGT-3’；
DCV-Taqman:5’-AGGACAAGAAGCCTGAC-3’；
JEV-F:5’-CGCGGACCAGGCATTC-3’；
JEV-R:5’-GAACTCTCCCCATGTGTTTGGA-3’；
JEV-Taqman:5’-AAGCGAAGCAGGAGAT-3’；
MHP-F:5’-TGACCGTCTTAACTCGCCAAAT-3’；
MHP-R:5’-GGCGGTAGGGAATTAAAACTATTG-3’；
MHP-Taqman:5’-CGCAAGTCTTGATAAAA-3’；
PRTV-F:5’-TTTGTTTACATTGACTACTGGGATGAT-3’；
PRTV-R:5’-AGCAGCAAGTGACCGAACATATAC-3’；
PRTV-Taqman:5’-ACAAGCATTCAGAAACA-3’；
CSFV-F：5’-TTTGCGTGGCAGGTTCCT-3’；
CSFV-R：5’-AATGATGCCAAATACCTCCTACTGA-3’；
CSFV-Taqman:5’-AAAGTCACAGCACTTAAT-3’；
SIV-F:5’-CCGAAATCGCGCAGAGA-3’；
SIV-R:5’-CATGAGAGCCTCAAGATCTGTGTT-3’；
SIV-Taqman:5’-TGAAGATGTCTTTGCTGGAA-3’；
PPV-F:5’-GCAGCTAACACAAGAAAAGGTTATCAC-3’；
PPV-R:5’-CCTGAGCTGGCCTAATTGCT-3’；
PPV-Taqman:5’-TAATAGCTACACAGAAGCAA-3’；
PCV-F:5’-TGAGCGGGAAAATGCAGAA-3’；
PCV-R:5’-CCAGGTGGCCCCACAA-3’；
PCV-Taqman:5’-TGGAAGACGAATGTACACG-3’；
PRV-F:5’-CGTCGTGAGCAGCATGAT-3’；
PRV-R:5’-CCATGATGACCAGCACGAT-3’；
PRV-Taqman:5’-CATCGGGATCCTGGC-3’；
APP-F:5’-GGTACCGGTCTTAGGTATCATTGAA-3’；
APP-R:5’-CTTCGTGGTGACCGCAATTT-3’；
APP-Taqman:5’-CGTGCATATCTGCC-3’；
SS-F:5’-TTCGAAGCAACGCGAAGAA-3’；
SS-R:5’-CCCATCTCTAGGGCGGTCAT-3’；
SS-Taqman:5’-CCAGGTCTTGACATCC-3’；
HPS-F:5’-GCGGCAGGCTTAACACATG-3’；
HPS-R:5’-CGTCAGCAAAGAAAGCAAGCT-3’；
HPS-Taqman:5’-TCGAACGGTAGCAGGGA-3’；
SC-F:5’-CACAGTCCGTTGTCGAGCTTTA-3’；
SC-R:5’-GCGGCTCTAGCACCAACAC-3’；
SC-Taqman:5’-CAGATACTGCCTTTGCTT-3’。
Wherein the 5' end of the nucleotide sequence of Taqman probes of foot-and-mouth disease virus, transmissible gastroenteritis virus, porcine Japanese encephalitis virus, porcine rotavirus, swine influenza virus, porcine circovirus, porcine actinobacillus pleuropneumoniae and haemophilus parasuis is marked with a FAM fluorescent reporter group; the 5' end of the nucleotide sequence of the Taqman probes of porcine epidemic diarrhea virus, porcine coronavirus, mycoplasma hyopneumoniae, swine fever virus, porcine parvovirus, porcine pseudorabies virus, streptococcus suis and chlamydia suis is marked with a ROX fluorescence report 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. Sample nucleic acid extraction
Foot and mouth disease virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine coronavirus, porcine Japanese encephalitis virus, mycoplasma hyopneumoniae, porcine rotavirus, swine fever virus, swine influenza virus, porcine parvovirus, porcine circovirus, porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae, streptococcus suis, haemophilus parasuis and porcine chlamydia nucleic acid are extracted using a total DNA/RNA extraction kit (EAR 005, beijing hundred bio-technology limited). Placing at-20deg.C for use.
3. Freeze-dried microchip preparation
The preparation of 8 groups of freeze-dried microchip RT-PCR systems, namely porcine foot-and-mouth disease virus and porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine coronavirus, porcine Japanese encephalitis virus and porcine mycoplasma pneumoniae, porcine rotavirus and swine fever virus, porcine influenza virus and porcine parvovirus, porcine circovirus and porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae and streptococcus suis, and haemophilus parasuis and chlamydia freeze-dried microchip RT-PCR systems, is carried out according to the following reaction systems:
the number of the whole freeze-dried microchip loading holes of the AriaYSB microchip (Beijing Yisen Biotechnology Co., ltd.) for fluorescence quantitative PCR was 24, and 2. Mu.L of each of the above fluorescence quantitative PCR positive control hole, sample hole and negative control Kong Donggan microchip RT-PCR system (comprising 2 kinds of primer probes) was added to the positive control hole, sample hole and negative control hole, respectively.
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
The lyophilized microchip was diluted with Taq Buffer (10×), with KCl, (Thermo Scientific, cat# B650060) and added nuclease-free water to dilute to 2 Xbuffer and 1 Xbuffer. Positive and negative control wells were added with 1 Xbuffer 2. Mu.L per well. Sample wells were added with 2 Xbuffer 1. Mu.L per well. 600 μl of mineral oil was added to the microchip surface, after which it covered all of the loading wells. The sample wells of 16 swine disease pathogen freeze-dried microchips were filled with 1 μl of foot-and-mouth disease virus, porcine epidemic diarrhea virus, swine transmissible gastroenteritis virus, porcine coronavirus, porcine Japanese encephalitis virus, porcine mycoplasma pneumoniae, porcine rotavirus, swine fever virus, porcine influenza virus, porcine parvovirus, porcine circovirus, porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae, porcine streptococcus, haemophilus parasuis, and porcine chlamydia acid extracts, respectively.
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 FAM and ROX channel fluorescence signal detection at the end of extension of each cycle.
The freeze-dried microchip is coated with the specificity experimental result of 16 swine disease pathogen dual fluorescent RT-PCR reagents. The specificity detection of the porcine foot-and-mouth disease virus is shown in figures 1 to 3, and the results of figure 1 show that the positive control of the porcine foot-and-mouth disease virus detected by the FAM channel and the RNA of the porcine foot-and-mouth disease virus are amplified, the Ct values are respectively 20.56 and 24.93, and the amplification curves are provided, and the negative control is not amplified. The results of FIG. 2 show that the positive control of the FAM channel for detecting the porcine foot-and-mouth disease virus is amplified, the Ct value is 20.07, and the RNA of the porcine epidemic diarrhea virus and the negative control are not amplified. The results of FIG. 3 show that the positive control for FAM channel detection of porcine foot and mouth disease virus has been amplified, with Ct value of 20.74, porcine transmissible gastroenteritis virus, porcine coronavirus, porcine Japanese encephalitis virus, porcine mycoplasma pneumoniae, porcine rotavirus, swine fever virus, swine influenza virus, porcine parvovirus, porcine circovirus, porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae, porcine streptococcus, haemophilus parasuis, porcine chlamydia, porcine blue ear virus and negative control have not been amplified. Specific detection of porcine epidemic diarrhea virus is shown in fig. 4-6, and the result of fig. 4 shows that the ROX channel detection of porcine epidemic diarrhea virus positive control and porcine transmissible gastroenteritis virus RNA is amplified, ct values are respectively 22.77 and 18.40, and amplification curves are provided, and the negative control is not amplified. The results of FIG. 5 show that the ROX channel detection porcine epidemic diarrhea virus positive control is amplified, the Ct value is 22.20, and the porcine foot and mouth disease virus RNA and the negative control are not amplified. The results of FIG. 6 show that the ROX channel detection of porcine epidemic diarrhea virus positive control was amplified, with Ct value 22.47, porcine transmissible gastroenteritis virus, porcine coronavirus, porcine Japanese encephalitis virus, porcine mycoplasma pneumoniae, porcine rotavirus, swine fever virus, swine influenza virus, porcine parvovirus, porcine circovirus, porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae, porcine streptococcus, haemophilus parasuis, porcine chlamydia, porcine blue ear virus and negative control were not amplified.
The amplification curve of the invention for specific detection of other 14 swine pathogens (transmissible gastroenteritis virus, swine coronavirus, swine Japanese encephalitis virus, mycoplasma hyopneumoniae, swine rotavirus, swine fever virus, swine influenza virus, swine parvovirus, porcine circovirus, porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae, swine streptococcus, haemophilus parasuis, and swine chlamydia pathogens) is basically identical to the form of the amplification curve of figures 1-6, so that the invention is not listed one by one for saving space. The specificity detection result of the swine transmissible gastroenteritis virus shows that the FAM channel is used for detecting the positive control of the swine transmissible gastroenteritis virus and the RNA of the swine transmissible gastroenteritis virus, the Ct value is 19.73, the Ct value is 24.00, and other pathogens and negative control are not amplified. The specificity result of the porcine epidemic diarrhea virus shows that the ROX channel detection porcine coronavirus positive control and the porcine coronavirus DNA are amplified, the Ct values are respectively 21.15 and 28.72, the amplification curves are respectively provided, and other pathogens and negative control are not amplified. The specificity detection result of the porcine Japanese encephalitis virus shows that the FAM channel is used for detecting positive control of the porcine Japanese encephalitis virus and RNA of the porcine Japanese encephalitis virus, the Ct value is 22.95 and the Ct value is 27.83 respectively, and amplification curves exist, and other pathogens and negative control are not amplified. The specific detection result of mycoplasma hyopneumoniae shows that the ROX channel detects positive control of mycoplasma hyopneumoniae and mycoplasma hyopneumoniae DNA and has amplification curves with Ct values of 21.64 and 27.42, and other pathogens and negative control have no amplification. The specificity detection result of the porcine rotavirus shows that the FAM channel detects positive control of the porcine rotavirus and RNA of the porcine rotavirus to be amplified, ct values are 19.76 and 21.58 respectively, amplification curves exist, and other pathogens and negative control are not amplified. The specificity detection result of the swine fever virus shows that the ROX channel detects the positive control of the swine fever virus and the RNA of the swine fever virus to be amplified, the Ct value is 19.51 and 23.89 respectively, the amplification curves are provided, and other pathogens and negative controls are not amplified. The specificity detection result of the swine influenza virus shows that positive control and RNA of the swine influenza virus detected by the FAM channel are amplified, ct values are 18.30 and 15.83 respectively, amplification curves are provided, and other pathogens and negative control are not amplified. The specificity detection result of the porcine parvovirus shows that the ROX channel detects the positive control of the porcine parvovirus and the DNA of the porcine parvovirus to be amplified, the Ct value is 17.39 and 16.09 respectively, the amplification curve exists, and other pathogens and negative controls are not amplified. The specificity detection result of the porcine circovirus shows that FAM channel detection porcine circovirus positive control and porcine circovirus DNA are amplified, ct values are 17.84 and 23.90 respectively, amplification curves are provided, and other pathogens and negative control are not amplified. The specificity detection result of the porcine pseudorabies virus shows that the ROX channel detects the positive control of the porcine pseudorabies virus and the DNA of the porcine pseudorabies virus to be amplified, the Ct values are 18.11 and 23.27 respectively, the amplification curves exist, and other pathogens and negative controls are not amplified. The specificity detection result of the actinobacillus pleuropneumoniae shows that FAM channel detection positive control and DNA of the actinobacillus pleuropneumoniae are amplified, ct values are 18.83 and 16.67 respectively, amplification curves exist, and other pathogens and negative control are not amplified. The specificity detection result of the streptococcus suis shows that the positive control and the streptococcus suis DNA of the ROX channel detection are amplified, the Ct value is 17.10 and 18.93 respectively, the amplification curves are provided, and other pathogens and negative controls are not amplified. The specificity detection result of haemophilus parasuis shows that FAM channel detection positive control and haemophilus parasuis DNA are amplified, ct values are 19.19 and 19.54 respectively, amplification curves exist, and other pathogens and negative control are not amplified. The specificity detection result of the swine chlamydia shows that the ROX channel detects the positive control of the swine chlamydia and the swine chlamydia DNA to be amplified, the Ct value is 20.10 and 26.06 respectively, the amplification curve is provided, and other pathogens and negative controls are not amplified. The results obtained are completely identical to the expected ones.
Experimental example 2, 16 swine disease pathogens freeze-dried microchip fluorescent RT-PCR detection kit and conventional fluorescent RT-PCR test Agent sensitivity validation and comparison
The concentration was 1X 10 7 TCID 50 The swine foot-and-mouth disease virus liquid/mL was subjected to 10-fold gradient dilution. Extraction of 1X 10 6 TCID 50 /mL～1×10 0 TCID 50 Genomic RNA of swine foot-and-mouth disease virus with each gradient concentration is taken as a template, and no nuclease water is taken as a negative pairAnd (3) performing universal sensitivity detection on the swine foot-and-mouth disease virus detected by using the freeze-dried microchip fluorescent RT-PCR reagent and the conventional fluorescent RT-PCR reagent.
The preparation of a freeze-dried microchip RT-PCR system A is carried out according to the following reaction system:
the number of the whole freeze-dried microchip loading holes of the AriaYSB microchip (Beijing Yisen Biotechnology Co., ltd.) for fluorescence quantitative PCR was 24, and 2. Mu.L of each of the above fluorescence quantitative PCR positive control hole, sample hole and negative control Kong Donggan microchip RT-PCR system (comprising 2 kinds of primer probes) was added to the positive control hole, sample hole and negative control hole, respectively.
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.
The whole lyophilized microchip loading wells of the AriaYSB microchip (pegjingesen biotechnology limited) for fluorescent quantitative PCR were 24, and the above-mentioned lyophilized microchip positive control wells and negative control wells (containing 2 primer probes for porcine foot-and-mouth disease virus and porcine epidemic diarrhea virus) were added to 2 μl of 1 x buffer. 600. Mu.L of mineral oil was added to the microchip surface and allowed to cover all of the wells. And 1×10 samples were added to the wells 6 TCID 50 /mL～1×10 0 TCID 50 Each gradient concentration of genomic RNA of porcine foot-and-mouth disease virus.
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 5 seconds and 60 ℃ for 15 seconds, 40 cycles are adopted in the third step, and FAM channel fluorescence signal detection is carried out at the extension end of each cycle in the third step.
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, at the end of extension of each cycle, FAM channel fluorescence signal detection was performed.
FIG. 7 shows that the concentration of the lowest detection sample is 1X 10 by using the lyophilized microchip fluorescent RT-PCR 1 TCID 50 The Ct value of the reaction solution is 31.56 per mL, so that the reaction cycle number 40 can greatly meet the minimum detection requirement. 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. 8 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 32.56 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. 9 and 10 show that the fluorescence RT-PCR amplification efficiency of the freeze-dried microchip is 118.78%, the fluorescence RT-PCR amplification efficiency of the conventional fluorescence RT-PCR is 98.88%, and the fluorescence RT-PCR amplification efficiency of the freeze-dried microchip is higher. The standard curve formulas and the correlation coefficients of the two are relatively close. The correlation coefficient of the fluorescence RT-PCR of the freeze-dried microchip is 0.998, and the correlation coefficient of the conventional fluorescence RT-PCR is 0.996, so that the correlation of the fluorescence RT-PCR of the freeze-dried microchip is better. The fluorescence RT-PCR reaction system of the freeze-dried microchip is 2 mu L, the conventional fluorescence RT-PCR system is 25 mu L, and the fluorescence RT-PCR reaction system of the freeze-dried microchip is reduced by more than 20 times. The running time of the fluorescent RT-PCR reaction of the freeze-dried microchip is less than 40 minutes, and the conventional fluorescent RT-PCR reaction time is 120 minutesThe running time is shortened by 2/3.
Experimental example 3: preparation and detection of 16 swine disease pathogen freeze-dried microchip fluorescent RT-PCR detection kit
1. Preparation of the kit:
a lyophilized microchip fluorescent RT-PCR system was prepared as in example 1.
Reagent 1: dilution Taq Buffer (10×) (Thermo Scientific, cat# B650060), 5 μl
Reagent 2: mineral oil (Sangon Biotech, cat. No. A630217) 1mL
Reagent 3: no nuclease 50 μl.
2. Repetitive analysis of kits
3 samples of known positives were selected and tested repeatedly between batches. Experiments were repeated between batches: 3 known positive samples were tested in batches, each sample being tested separately.
Reagent 1 was diluted to 2 Xbuffer and 1 Xbuffer with reagent 3 before use, and 1 Xbuffer 2. Mu.L was added to each of the positive control and negative control wells; 600. Mu.L of reagent 2 was added to the microchip surface and allowed to cover all of the wells. Sample wells 2. Mu.L of sample wells per well was added after mixing 2 Xbuffer and sample nucleic acid at 1:1.
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.
From the test results in Table 1, it can be seen that the variation coefficient between batches is between 0.25% and 1.95%, which indicates that the kit has good reproducibility.
Table 1 kit reproducibility assay
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By adopting the technical scheme, the invention develops a reaction system for detecting 16 pig disease pathogens rapidly and effectively by designing specific primers and probes and optimizing microchip freeze-drying conditions, and simultaneously prepares a detection kit based on the method, wherein the detection method has high accuracy, specificity and sensitivity and good 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 16 swine pathogens
<130> P180497/YSB
<160> 48
<170> PatentIn version 3.5
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<223> FMDV-F
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ggccgagacc acaaatgtg 19
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tcggccttgc catgtgtaa 19
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aggacaagaa gcctgac 17
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gaactctccc catgtgtttg ga 22
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aagcgaagca ggagat 16
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tgaccgtctt aactcgccaa at 22
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ggcggtaggg aattaaaact attg 24
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cgcaagtctt gataaaa 17
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tttgtttaca ttgactactg ggatgat 27
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agcagcaagt gaccgaacat atac 24
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ccgaaatcgc gcagaga 17
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catgagagcc tcaagatctg tgtt 24
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tgaagatgtc tttgctggaa 20
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gcagctaaca caagaaaagg ttatcac 27
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cctgagctgg cctaattgct 20
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taatagctac acagaagcaa 20
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ggtaccggtc ttaggtatca ttgaa 25
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cttcgtggtg accgcaattt 20
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ttcgaagcaa cgcgaagaa 19
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cccatctcta gggcggtcat 20
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ccaggtcttg acatcc 16
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cacagtccgt tgtcgagctt ta 22
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cagatactgc ctttgctt 18

Claims (18)

1. A lyophilized microchip for identifying 16 swine pathogens, wherein a fluorescent PCR reaction system for identifying 16 swine pathogens is immobilized on the microchip by lyophilization;
the fluorescent PCR reaction system comprises: the following primers and probes:
FMDV-F:5’-GGCCGAGACCACAAATGTG-3’；
FMDV-R:5’-TCGGCCTTGCCATGTGTAA-3’；
FMDV-Taqman:5’-AGGGATGGGTCTGCTT-3’；
PEDV-F:5’-GCAAATACATTGGCAGCATAACC-3’；
PEDV-R:5’-TGCTGGTGAGGATGGTCTTTC-3’；
PEDV-Taqman:5’-ATGCAATTAGCTGTACAGTGT-3’；
TEGV-F:5’-TGGTGAGCCCTTGCAAATG-3’；
TEGV-R:5’-CAGTTACAACAACCCGCAATTTT-3’；
TEGV-Taqman:5’-TAATAGCACCTTCAGCAGAAT-3’；
DCV-F:5’-AGCCACCCACCAAACCAA-3’；
DCV-R:5’-TGGGTTTAGCAGACTGGTCTTGT-3’；
DCV-Taqman:5’-AGGACAAGAAGCCTGAC-3’；
JEV-F:5’-CGCGGACCAGGCATTC-3’；
JEV-R:5’-GAACTCTCCCCATGTGTTTGGA-3’；
JEV-Taqman:5’-AAGCGAAGCAGGAGAT-3’；
MHP-F:5’-TGACCGTCTTAACTCGCCAAAT-3’；
MHP-R:5’-GGCGGTAGGGAATTAAAACTATTG-3’；
MHP-Taqman:5’-CGCAAGTCTTGATAAAA-3’；
PRTV-F:5’-TTTGTTTACATTGACTACTGGGATGAT-3’；
PRTV-R:5’-AGCAGCAAGTGACCGAACATATAC-3’；
PRTV-Taqman:5’-ACAAGCATTCAGAAACA-3’；
CSFV-F：5’-TTTGCGTGGCAGGTTCCT-3’；
CSFV-R：5’-AATGATGCCAAATACCTCCTACTGA-3’；
CSFV-Taqman:5’-AAAGTCACAGCACTTAAT-3’；
SIV-F:5’-CCGAAATCGCGCAGAGA-3’；
SIV-R:5’-CATGAGAGCCTCAAGATCTGTGTT-3’；
SIV-Taqman:5’-TGAAGATGTCTTTGCTGGAA-3’；
PPV-F:5’-GCAGCTAACACAAGAAAAGGTTATCAC-3’；
PPV-R:5’-CCTGAGCTGGCCTAATTGCT-3’；
PPV-Taqman:5’-TAATAGCTACACAGAAGCAA-3’；
PCV-F:5’-TGAGCGGGAAAATGCAGAA-3’；
PCV-R:5’-CCAGGTGGCCCCACAA-3’；
PCV-Taqman:5’-TGGAAGACGAATGTACACG-3’；
PRV-F:5’-CGTCGTGAGCAGCATGAT-3’；
PRV-R:5’-CCATGATGACCAGCACGAT-3’；
PRV-Taqman:5’-CATCGGGATCCTGGC-3’；
APP-F:5’-GGTACCGGTCTTAGGTATCATTGAA-3’；
APP-R:5’-CTTCGTGGTGACCGCAATTT-3’；
APP-Taqman:5’-CGTGCATATCTGCC-3’；
SS-F:5’-TTCGAAGCAACGCGAAGAA-3’；
SS-R:5’-CCCATCTCTAGGGCGGTCAT-3’；
SS-Taqman:5’-CCAGGTCTTGACATCC-3’；
HPS-F:5’-GCGGCAGGCTTAACACATG-3’；
HPS-R:5’-CGTCAGCAAAGAAAGCAAGCT-3’；
HPS-Taqman:5’-TCGAACGGTAGCAGGGA-3’；
SC-F:5’-CACAGTCCGTTGTCGAGCTTTA-3’；
SC-R:5’-GCGGCTCTAGCACCAACAC-3’；
SC-Taqman:5’-CAGATACTGCCTTTGCTT-3’；
the lowest detection sample concentration of the freeze-dried microchip for identifying 16 swine pathogens is 1 multiplied by 10 1 TCID 50 /mL；
The 16 swine pathogens include: porcine foot and mouth disease virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine coronavirus, porcine Japanese encephalitis virus, mycoplasma hyopneumoniae, porcine rotavirus, swine fever virus, swine influenza virus, porcine parvovirus, porcine circovirus, porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae, porcine streptococcus, haemophilus parasuis, and porcine chlamydia.
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 probe of porcine foot-and-mouth disease virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine coronavirus, porcine encephalitis b virus, mycoplasma hyopneumoniae, porcine rotavirus, swine fever virus, swine influenza virus, porcine parvovirus, porcine circovirus, porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae, porcine streptococcus, haemophilus parasuis and chlamydia 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 or 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; dNTP 0.3mM; mg of 2+ 3mM; trehalose 5 μm; tris-Cl 5mM, template 5 ng/. Mu.L balance sterile deionized water.
5. The lyophilized microchip according to claim 4, wherein the template is selected from the group consisting of cDNA of 16 swine pathogens, cDNA of a sample to be tested and sterilized deionized water, which are used as positive control, sample detection system and negative control, respectively.
6. The lyophilized microchip according to any one of claims 1-3, 5, wherein the upstream primer, the downstream primer and the Taqman probe of the fluorescent PCR reaction system in each well before the microchip is lyophilized respectively comprise a primer set and a Taqman probe of 2 swine pathogens out of 16 swine pathogens, and the template of the fluorescent PCR reaction system of the positive control is respectively a cDNA mixture of 2 swine pathogens out of 16 swine pathogens; the 2 swine pathogens are selected from: foot and mouth disease virus and porcine epidemic diarrhea virus; transmissible gastroenteritis virus and porcine coronavirus; encephalitis b virus and mycoplasma hyopneumoniae; porcine rotavirus and swine fever virus; swine influenza virus and swine parvovirus; porcine circovirus and porcine pseudorabies virus; actinobacillus pleuropneumoniae and streptococcus suis; or haemophilus parasuis and chlamydia suis.
7. The lyophilized microchip according to claim 6, wherein the 5' -end-labeled fluorescent reporter group of Taqman probes of porcine foot-and-mouth disease virus, transmissible gastroenteritis virus, porcine encephalitis b virus, porcine rotavirus, porcine influenza virus, porcine circovirus, porcine actinobacillus pleuropneumoniae and haemophilus parasuis is a FAM fluorescent reporter group;
the fluorescent reporter group marked at the 5' end of the Taqman probes of the porcine epidemic diarrhea virus, porcine coronavirus, mycoplasma hyopneumoniae, swine fever virus, porcine parvovirus, porcine pseudorabies virus, streptococcus suis and chlamydia suis is a ROX fluorescent reporter group;
the Taqman probe 3' -end-labeled fluorescence quenching group of the porcine foot-and-mouth disease virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine coronavirus, porcine Japanese encephalitis virus, porcine mycoplasma pneumoniae, porcine rotavirus, swine fever virus, porcine influenza virus, porcine parvovirus, porcine circovirus, porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae, porcine streptococcus, haemophilus parasuis and porcine chlamydia is an MGB quenching group.
8. The lyophilized microchip according to any one of claims 1-3, 5, 7, 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.
9. The lyophilized microchip according to claim 8, wherein the number of loading wells on the microchip is 24; the structure of the microchip is matched with the structure of a sample adding plate of the PCR instrument.
10. The lyophilized microchip according to any one of claims 1-3, 5, 7, 9, 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.
11. The lyophilized microchip according to claim 10, 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.
12. A kit for identifying 16 swine pathogens comprising the lyophilized microchip of any one of claims 1-11; the 16 swine pathogens include: porcine foot and mouth disease virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine coronavirus, porcine Japanese encephalitis virus, mycoplasma hyopneumoniae, porcine rotavirus, swine fever virus, swine influenza virus, porcine parvovirus, porcine circovirus, porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae, porcine streptococcus, haemophilus parasuis, and porcine chlamydia.
13. The kit of claim 12, 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.
14. The kit of claim 12 or 13, further comprising: mineral oil, closing the loading wells on the lyophilized microchip.
15. A method for identifying 16 swine pathogens for non-diagnostic purposes, characterized in that a lyophilized microchip according to any one of claims 1-11 and/or a kit according to any one of claims 12-14 is used for fluorescent PCR amplification of a sample to be tested; the 16 swine pathogens include: porcine foot and mouth disease virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine coronavirus, porcine Japanese encephalitis virus, mycoplasma hyopneumoniae, porcine rotavirus, swine fever virus, swine influenza virus, porcine parvovirus, porcine circovirus, porcine pseudorabies virus, porcine actinobacillus pleuropneumoniae, porcine streptococcus, haemophilus parasuis, and porcine chlamydia.
16. The method of claim 15, 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.
17. The method of claim 15, wherein the diluent is 10 x buffer.
18. 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.
CN201811053108.1A 2018-09-10 2018-09-10 Freeze-dried microchip, kit and method for identifying 16 pig disease pathogens Active CN110885904B (en)

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