CN111500767A - Microfluid chip for simultaneously detecting 9 porcine pathogens - Google Patents
Microfluid chip for simultaneously detecting 9 porcine pathogens Download PDFInfo
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Abstract
The invention provides a microfluidic chip for simultaneously detecting 9 swine pathogens, which belongs to the technical field of genetic engineering and is characterized in that a swine fever virus primer probe, a porcine pseudorabies virus primer probe, a porcine ridge virus primer probe, a porcine parvovirus I-type primer probe, a porcine parvovirus IV-type primer probe, a porcine epidemic encephalitis B virus primer probe, a American porcine reproductive and respiratory syndrome virus primer probe, a highly pathogenic porcine reproductive and respiratory syndrome virus primer probe and a porcine circovirus 2-type primer probe are coated on the microfluidic chip. Sensitivity of the chip: 10 copies of classical swine fever virus, 100 copies of porcine pseudorabies virus, 100 copies of porcine kobuvirus, 100 copies of porcine parvovirus I, 100 copies of porcine parvovirus IV, 100 copies of porcine encephalitis B virus, 100 copies of porcine circovirus 2, 100 copies of highly pathogenic porcine reproductive and respiratory syndrome virus and 10 copies of American porcine reproductive and respiratory syndrome virus.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a microfluid chip for simultaneously detecting 9 porcine pathogens.
Background
The swine fever virus, the porcine parvovirus, the porcine pseudorabies virus, the porcine circovirus, the porcine reproductive and respiratory syndrome virus, the highly pathogenic porcine reproductive and respiratory syndrome virus, the porcine epidemic encephalitis B virus and other 9 pathogens seriously harm the pig industry, the differential diagnosis of the porcine pathogens is rapidly and accurately carried out, the prevention and the control of animal epidemic diseases are facilitated, and the positive significance is realized on the healthy development of the animal industry.
At present, laboratory methods for hog cholera virus, porcine parvovirus, porcine rabies virus, porcine circovirus, porcine reproductive and respiratory virus, highly pathogenic porcine reproductive and respiratory virus and porcine epidemic encephalitis B virus are mainly PCR and fluorescent quantitative PCR methods, although the detection speed, sensitivity and specificity of the PCR and the fluorescent quantitative PCR methods are obviously improved compared with the traditional virus culture and antigen detection methods, in view of the fact that viruses are various, the PCR and fluorescent quantitative PCR detection methods in the current clinical application can only detect single or less than three porcine viruses at a time, cannot screen the plurality of porcine viruses at the same time, and can be definite after multiple fluorescent quantitative PCR or PCR methods are used for detecting the porcine pathogens to screen, time and labor are wasted, and samples are wasted.
Disclosure of Invention
In view of this, the present invention provides a microfluidic chip for simultaneously detecting 9 porcine pathogens, and the microfluidic chip provided by the present invention can simultaneously detect 9 porcine pathogens.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a microfluidic chip for simultaneously detecting 9 swine pathogens, which is characterized in that a swine fever virus primer probe, a porcine pseudorabies virus primer probe, a porcine kobuvirus primer probe, a porcine parvovirus I-type primer probe, a porcine parvovirus IV-type primer probe, a porcine epidemic encephalitis B virus primer probe, a American porcine reproductive and respiratory syndrome virus primer probe, a highly pathogenic porcine reproductive and respiratory syndrome virus primer probe and a porcine circovirus 2-type primer probe are coated on the microfluidic chip;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the primer probe for swine fever virus are shown as SEQ ID No. 1-3 in sequence;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the porcine pseudorabies virus primer probe are shown as SEQ ID No. 4-6 in sequence;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the porcine kobuvirus primer probe are sequentially shown as SEQ ID Nos. 7-9;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the porcine parvovirus type I primer probe are shown as SEQ ID No. 10-12 in sequence;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the porcine parvovirus type IV primer probe are shown as SEQ ID No. 13-15 in sequence;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the swine epidemic encephalitis B virus primer probe are sequentially shown as SEQ ID No. 16-18;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the American porcine reproductive and respiratory syndrome virus primer probe are sequentially shown in SEQ ID Nos. 19-21;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the highly pathogenic porcine reproductive and respiratory syndrome virus primer probe are sequentially shown as SEQ ID No. 22-24;
the nucleotide sequences of the upstream primer, the downstream primer and the probe of the porcine circovirus type 2 primer probe are shown as SEQ ID No. 25-27 in sequence.
Preferably, FAM is added to the 5 'end of the probe of the swine fever virus primer probe, and QSY1 is added to the 3' end of the probe.
Preferably, FAM is added to the 5 'end of the probe of the porcine pseudorabies virus primer probe, and QSY1 is added to the 3' end of the probe.
Preferably, FAM is added to the 5 'end of the probe of the porcine kobuvirus primer probe, and QSY1 is added to the 3' end of the probe.
Preferably, FAM is added to the 5 'end of the probe of the porcine parvovirus type I primer probe, and QSY1 is added to the 3' end of the probe.
Preferably, FAM is added to the 5 'end of the probe of the porcine parvovirus type IV primer probe, and QSY1 is added to the 3' end of the probe.
Preferably, FAM is added to the 5 'end of the probe of the swine epidemic encephalitis B virus primer probe, and QSY1 is added to the 3' end of the probe.
Preferably, FAM is added to the 5 'end of the probe of the American porcine reproductive and respiratory syndrome virus primer probe, and QSY1 is added to the 3' end of the probe.
Preferably, FAM is added to the 5 'end of the probe of the highly pathogenic porcine reproductive and respiratory syndrome virus primer probe, and QSY1 is added to the 3' end of the probe.
Preferably, FAM is added to the 5 'end of the probe of the porcine circovirus type 2 primer probe, and QSY1 is added to the 3' end of the probe.
The invention provides a microfluidic chip for simultaneously detecting 9 swine pathogens, which is characterized in that a swine fever virus primer probe, a porcine pseudorabies virus primer probe, a porcine crest virus primer probe, a porcine parvovirus I-type primer probe, a porcine parvovirus IV-type primer probe, a porcine epidemic encephalitis B virus primer probe, a American porcine reproductive and respiratory syndrome virus primer probe, a highly pathogenic porcine reproductive and respiratory syndrome virus primer probe and a porcine circovirus 2-type primer probe are coated on the microfluidic chip.
The detection sensitivity of the microfluidic chip provided by the invention is as follows: 10 copies of classical swine fever virus, 100 copies of porcine pseudorabies virus, 100 copies of porcine kobuvirus, 100 copies of porcine parvovirus I, 100 copies of porcine parvovirus IV, 100 copies of porcine epidemic encephalitis B virus, 100 copies of porcine circovirus 2, 100 copies of porcine highly pathogenic reproductive and respiratory syndrome virus and 10 copies of American porcine reproductive and respiratory syndrome virus.
Drawings
FIG. 1 is a classical swine fever virus microfluidic chip amplification standard curve;
FIG. 2 is a standard amplification curve of a porcine pseudorabies virus microfluidic chip;
FIG. 3 is a standard amplification curve of a porcine kobuvirus microfluidic chip;
FIG. 4 is a standard amplification curve for a porcine parvovirus type I microfluidic chip;
FIG. 5 is a standard amplification curve for a porcine type IV parvovirus microfluidic chip;
FIG. 6 is a standard amplification curve of a swine Japanese encephalitis virus microfluidic chip;
FIG. 7 is a standard amplification curve for porcine circovirus type 2 microfluidic chip;
FIG. 8 is a standard amplification curve of a highly pathogenic PRRSV microfluidic chip;
FIG. 9 is a standard curve for amplification of a microfluidic chip for porcine reproductive and respiratory syndrome virus of the American type.
Detailed Description
The invention provides a microfluidic chip for simultaneously detecting 9 swine pathogens, which is characterized in that a swine fever virus primer probe, a porcine pseudorabies virus primer probe, a porcine crest virus primer probe, a porcine parvovirus I-type primer probe, a porcine parvovirus IV-type primer probe, a porcine epidemic encephalitis B virus primer probe, a American porcine reproductive and respiratory syndrome virus primer probe, a highly pathogenic porcine reproductive and respiratory syndrome virus primer probe and a porcine circovirus 2-type primer probe are coated on the microfluidic chip.
In the invention, the nucleotide sequences of an upstream primer, a downstream primer and a probe of the primer probe for classical swine fever virus are shown as SEQ ID No. 1-3 in sequence, and specifically as follows:
SEQ ID No.1:5-ATGCCCAYAGTAGGACTAGCA-3；
SEQ ID No.2:5-CTACTGACGACTGTCCTGTAC-3；
in SEQ ID No.3:5-TGGCGAGCTCCCTGGGTGGTCTAAGT-3, it is preferable that FAM is added to the 5 'end of the probe and QSY1 is added to the 3' end of the probe.
In the invention, the nucleotide sequences of an upstream primer, a downstream primer and a probe of the porcine pseudorabies virus primer probe are shown as SEQ ID No. 4-6 in sequence, and specifically as follows:
SEQ ID No.4:5-ACAAGTTCAAGGCCCACATCTAC-3；
SEQ ID No.5:5-GTCYGTGAAGCGGTTCGTGAT-3；
in SEQ ID No.6:5-ACGTCATCGTCACGACC-3, it is preferable that FAM is added to the 5 'end of the probe and QSY1 is added to the 3' end of the probe.
In the invention, the nucleotide sequences of an upstream primer, a downstream primer and a probe of the porcine kobuvirus primer probe are shown as SEQ ID No. 7-9 in sequence, and specifically as follows:
SEQ ID No.7:5-CACYCGYGTCAGAGATCAGGT-3；
SEQ ID No.8:5-AGTCTATTCTACACARRGCGATACAGAGC-3；
SEQ ID No.9:5-TGTATCTGGTGTCACGCTGCCA-3, preferably FAM is added to the 5 'end of the probe and QSY1 is added to the 3' end of the probe when in use.
In the invention, the nucleotide sequences of an upstream primer, a downstream primer and a probe of the porcine parvovirus type I primer probe are shown as SEQ ID No. 10-12 in sequence, and specifically as follows:
SEQ ID No.10:5-GAAGACTGGATGATGACAGATCCA-3；
SEQ ID No.11:5-TGCTGTTTTTGTTCTTGCTAGAGTAA-3；
in SEQ ID No.12:5-AATGATGGCTCAAACCGGAGGAGA-3, it is preferable that FAM is added to the 5 'end of the probe and QSY1 is added to the 3' end of the probe.
In the invention, the nucleotide sequences of an upstream primer, a downstream primer and a probe of the porcine parvovirus IV-type primer probe are shown as SEQ ID No. 13-15 in sequence, and are specifically shown as follows:
SEQ ID No.13:5-TTTGCCAATAGTGCACAAGG-3；
SEQ ID No.14:5-TGAGGCATCCATGGGTCTATCA-3；
in SEQ ID No.15:5-CAGAAAGCAAACTGAGATGTCC-3, it is preferable that FAM is added to the 5 'end of the probe and QSY1 is added to the 3' end of the probe.
In the invention, the nucleotide sequences of an upstream primer, a downstream primer and a probe of the swine epidemic encephalitis B virus primer probe are shown as SEQ ID Nos. 16-18 in sequence, and are specifically shown as follows:
SEQ ID No.16:5-GGCTCTTATCACGTTCTTCAAGTTT-3；
SEQ ID No.17:5-TGCTTTCCATCGGCCYAAAA-3；
SEQ ID No.18:5-AGCATTAGCCCCGACCAAGGCG-3, preferably FAM is added to the 5 'end of the probe and QSY1 is added to the 3' end of the probe when in use.
In the invention, the nucleotide sequences of an upstream primer, a downstream primer and a probe of the American porcine reproductive and respiratory syndrome virus primer probe are shown as SEQ ID Nos. 19-21 in sequence, and are specifically shown as follows:
SEQ ID No.19:5-ATRATGRGCTGGCATTC-3；
SEQ ID No.20:5-ACACGGTCGCCCTAATTG-3；
SEQ ID No.21:5-TGTGGTGAATGGCACTGATTGACA-3, preferably FAM is added to the 5 'end of the probe and QSY1 is added to the 3' end of the probe when in use.
In the invention, the nucleotide sequences of an upstream primer, a downstream primer and a probe of the highly pathogenic porcine reproductive and respiratory syndrome virus primer probe are shown as SEQ ID No. 22-24 in sequence, and specifically as follows:
SEQ ID No.22:5-CCGCGTAGAACTGTGACAAC-3；
SEQ ID No.23:5-TCCAGGATGCCCATGTTCTG-3；
SEQ ID No.24:5-ACGCACCAGGATGAGCCTCTGGAT-3, preferably FAM is added to the 5 'end of the probe and QSY1 is added to the 3' end of the probe when in use.
In the invention, the nucleotide sequences of an upstream primer, a downstream primer and a probe of the porcine circovirus type 2 primer probe are shown as SEQ ID No. 25-27 in sequence, and are specifically shown as follows:
SEQ ID No.25:5-CAGCACCCTGTAACGTTTGTC-3；
SEQ ID No.26:5-TTAGTCTTCCAATCACGCTTCTG-3；
SEQ ID No.27:5-TTTCCCGCTCACTTTCAAAAGTTCAGC-3, preferably FAM is added to the 5 'end of the probe and QSY1 is added to the 3' end of the probe when in use.
In the invention, the microfluidic chip is preferably further coated with an internal reference primer probe, and the nucleotide sequences of an upstream primer, a downstream primer and a probe of the internal reference primer probe are sequentially shown as SEQ ID No. 28-30, and specifically as follows:
SEQ ID No.28:5-GACCACTACCAGCAGAACAC-3；
SEQ ID No.29:5-GAACTCCAGCAGGACCATG-3；
SEQ ID No.30:5-AGCACCCAGTCCGCCCTGAGCA-3, preferably FAM is added to the 5 'end of the probe and QSY1 is added to the 3' end of the probe when in use.
In the present invention, the method of using the microfluidic chip preferably includes: adding 100 mul of reaction system into the sample adding hole of the microfluid chip, centrifuging, reacting in a V7 fluorescent quantitative PCR instrument, and collecting an amplification signal. In the present invention, the rotation speed of the centrifugation is preferably 1000rpm, the time of the centrifugation is preferably 2min, and the number of times of the centrifugation is preferably 2 times.
In The present invention, each 100. mu.l of The reaction system preferably comprises 30. mu.l of The nucleic acid template, 50. mu.l of The Ag-Path-ID one-step RT-PCR buffer, 4. mu.l of The enzyme mixture and 16. mu.l of RNase-free water, The above-mentioned reagents preferably being derived from The Ag-Path-ID one-step RT-PCR kit. The preparation method of the nucleic acid template is not particularly limited, and the nucleic acid template can be prepared by adopting a conventional method for extracting viral nucleic acid. In the present invention, the 100. mu.l reaction system is added into the loading well of the microfluidic chip, the final concentration of each probe is 250nM, and the final concentration of each primer is 600 nM.
In the present invention, the reaction conditions are preferably as follows: 20min at 45 ℃; 10min at 95 ℃; 95 ℃ for 15s, 60 ℃ for 1min, 45 cycles. The fluorescence signal was collected at 60 ℃.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Experimental Material
1.1 Virus and Positive samples
The high-concentration nucleic acid positive specimens of the porcine transmissible gastroenteritis virus, the porcine epidemic diarrhea virus, the porcine epidemic encephalitis B virus, the porcine pseudorabies virus, the hog cholera virus, the porcine parvovirus, the porcine kobuvirus, the porcine reproductive and respiratory syndrome virus and the like can be prepared by adopting a conventional method.
1.2 reagents
Virus nucleic acid extraction kit MagMAXTMThe-96 Viral RNA Isolation Kit was purchased from ABI, USA. One Step PrimeScriptTMRT-PCR Kit(Perfect Real Time)，pMDTM18-T Vector cloning Kit, Plasmid Extraction Kit MiniBEST Plasmid purification Kit, DNA Gel recovery Kit MiniBESTAgarose Gel DNA Extraction Kit, Ex Taq enzyme, D L2000 DNA Marker, DH5 α competent cells are all products of Dalianbao bioengineering Co., Ltd.
2. Experimental methods
2.1 primer design and preparation of microfluidic chips
Primers and probes were synthesized and coated on a microfluidic chip (table 1) by ABI in the united states to prepare a swine-associated virus microfluidic chip. The final concentrations of probes and primers when reacted in the microfluidic chip were 250nM and 600nM, respectively.
TABLE 1 primers and probes coated in individual reaction wells of a microfluidic chip
2.2 extraction of viral nucleic acids
Using viral nucleiAcid extraction kit MagMAXTMThe Viral nucleic acid was extracted from the-96 Viral RNA Isolation Kit according to the protocol and stored in a freezer at-80 ℃.
2.3 products of the above porcine virus recombinant plasmid standards
According to the target gene fragments detected in the above-mentioned various fluorescent quantitative detection methods, primers (Table 2) are designed to amplify the nucleic acid fragments containing the above-mentioned virus detection genes, specifically, various virus nucleic acids are used as templates, and One StepPrimeScript RT-PCR Kit is used to perform RT-PCR reaction, the reaction system is 2 × One step RT-PCR buffer 25. mu.l, Takara Ex Taq HS (5U/. mu.l) 1. mu.l, PrimeScript RT Enzyme Mix 1. mu.l, upstream primer P1 (20. mu. mol/L) 1. mu.l, downstream primer P2 (20. mu. mol/L) 1. mu.l, RNA 5. mu.l, RNA-free water 16. mu.l, the total volume is 50. mu.l, the reaction conditions are 42 ℃, 10min, 95 ℃ 10s, 95 ℃ 5s, 52 ℃ 30s, 72 ℃, 40s, 35 cycles, 72 ℃ sequencing 10min, the amplified various virus fragments are cloned in a target gene amplification vector constructed by using a limited light engineering technology, and the recombinant plasmid containing the target DNA constructed by a limited organism 18 constructed by using a limited light recombination technology.
TABLE 2 primers for amplifying nucleic acid fragments containing the genes detected by the above-mentioned fluorescent quantitation method
2.4 detection of microfluidic chips
The Ag-Path-ID one-step RT-PCR kit is used for operation. The reaction conditions were as follows: 45 cycles at 45 ℃ for 20min, 95 ℃ for 10min, (95 ℃ for 15sec, 60 ℃ for 1 min). The reaction system of each run was as follows: mu.l of nucleic acid template, 50. mu.l of lAg-Path-ID one-step RT-PCR buffer, 4. mu.l of enzyme mixture, 16. mu.l of RNase-free water. Add 100. mu.l of the reaction into the well of the microfluidic chip, centrifuge at 1000rpm for 1min, and repeat 2 times. And reacted in the V7 fluorescent quantitative PCR according to the above reaction conditions and the related amplification signals were collected.
2.5 evaluation of sensitivity of microfluidic chip and establishment of Standard curves for various viruses
At a final concentration of 10 in the reaction system7And (3) taking the recombinant plasmid with the copy/mul-1 copy/mul as a template, detecting by using the established fluorescent quantitative PCR method, and evaluating the sensitivity of the recombinant plasmid. And drawing a standard curve for the detection result by taking the logarithm and Ct value of the final concentration of the recombinant plasmid in the reaction system as an X axis and a Y axis respectively.
2.6 testing of clinical samples
The prepared microfluid chip is used for detecting 40 parts of pig disease materials, and compared with other methods, the microfluid chip detection method is comprehensively evaluated.
3 results and analysis
3.1 sensitivity of microfluidic chips and establishment of Standard Curve
The 9 swine pathogen detection primers and probes were loaded into microfluidic wells, and the sensitivity was determined using recombinant plasmids of various viruses, with the following results: 10 copies of classical swine fever virus, 100 copies of porcine pseudorabies virus, 100 copies of porcine kobuvirus, 100 copies of porcine parvovirus I, 100 copies of porcine parvovirus IV, 100 copies of porcine epidemic encephalitis B virus, 100 copies of porcine circovirus type 2, 100 copies of highly pathogenic porcine reproductive and respiratory syndrome virus, 10 copies of American porcine reproductive and respiratory syndrome virus, and 10 copies of various viruses7There was a good correlation between the concentrations detected to the lowest (see figures 1 to 9 for standard curves).
3.2 evaluation of specificity
Through detection and verification by a single fluorescent quantitative PCR method, when the primer probe coated by the microfluidic chip reacts, an amplification curve can appear with target viruses, and no cross reaction occurs with other viruses.
3.3 evaluation of clinical applications
The method comprises the steps of detecting 40 pig samples by using a microfluidic chip, and simultaneously detecting CSFV, PRV, PKOV, PRRSV, HPRRSV, PPV1, PPV4, JEV and PCV2 of the samples by using a single-fold fluorescent quantitative PCR method. The results show that the detection results of the microfluidic chip are highly consistent with those of the single fluorescent quantitative PCR method, but the detection results of the suspected sample with low virus content are different (Table 3).
Comparison of microfluidic chip (TAC) of the viruses of Table 39 with the fluorescent quantitative RT-PCR method
Therefore, the defect of low detection flux of the conventional fluorescent quantitative PCR and PCR method for detecting the swine pathogens is overcome, the 9 swine pathogen high-flux rapid detection method is established by combining the fluorescent quantitative PCR detection method and the microfluid chip technology, the primer probes aiming at various swine pathogens are embedded on one fluorescent quantitative PCR reaction chip, and the various swine pathogens can be detected by one-time reaction through the fluorescent quantitative PCR detection. The high sensitivity and specificity of the fluorescent quantitative PCR method are continued; the high-flux detection of pathogens can be realized, and the defect of low flux of the current PCR and fluorescent quantitative PCR detection method is overcome; the time consumption is short, the automation degree is high, and the pollution rate is low; and simultaneously, the required amount of sample nucleic acid and the consumed amount of reagents are greatly reduced.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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<210>25
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
cagcaccctg taacgtttgt c 21
<210>26
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
ttagtcttcc aatcacgctt ctg 23
<210>27
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
tttcccgctc actttcaaaa gttcagc 27
<210>28
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
<210>29
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
gaactccagc aggaccatg 19
<210>30
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
agcacccagt ccgccctgag ca 22
<210>31
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
acaagttcaa ggcccacatc tac 23
<210>32
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
<210>33
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
gaagactgga tgatgacaga tcca 24
<210>34
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
ttacaggagt tggttcaatt tg 22
<210>35
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
atgcccayag taggactagc a 21
<210>36
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
tcaactccat gtgccatgta c 21
<210>37
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
<210>38
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
acaaggcaaa cgttcctcca c 21
<210>39
<211>17
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
ggctccacta cggtcaa 17
<210>40
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
acacggtcgc cctaattg 18
<210>41
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
gtactctcta agttggaaga ggt 23
<210>42
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
<210>43
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
ggctcttatc acgttcttca agttt 25
<210>44
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
aaccagggtt ccttatgatc ca 22
<210>45
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>45
cagcaccctg taacgtttgt c 21
<210>46
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
taaccatgta tgtacaattc agag 24
<210>47
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
cgttggtttg tgcctgatga cat 23
<210>48
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
ggaraactcc accatctatg acgtc 25
Claims (10)
1. A microfluid chip for detecting 9 porcine pathogens simultaneously is characterized in that a swine fever virus primer probe, a porcine pseudorabies virus primer probe, a porcine crest virus primer probe, a porcine parvovirus I type primer probe, a porcine parvovirus IV type primer probe, a porcine epidemic encephalitis B virus primer probe, a American porcine reproductive and respiratory syndrome virus primer probe, a highly pathogenic porcine reproductive and respiratory syndrome virus primer probe and a porcine circovirus 2 type primer probe are coated on the microfluid chip;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the primer probe for swine fever virus are shown as SEQ ID No. 1-3 in sequence;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the porcine pseudorabies virus primer probe are shown as SEQ ID Nos. 4-6 in sequence;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the porcine kobuvirus primer probe are sequentially shown as SEQ ID Nos. 7-9;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the porcine parvovirus type I primer probe are sequentially shown as SEQID No. 10-12;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the porcine parvovirus type IV primer probe are sequentially shown as SEQID No. 13-15;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the swine epidemic encephalitis B virus primer probe are sequentially shown as SEQ ID No. 16-18;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the American porcine reproductive and respiratory syndrome virus primer probe are sequentially shown in SEQ ID Nos. 19-21;
the nucleotide sequences of an upstream primer, a downstream primer and a probe of the highly pathogenic porcine reproductive and respiratory syndrome virus primer probe are sequentially shown as SEQ ID No. 22-24;
the nucleotide sequences of the upstream primer, the downstream primer and the probe of the porcine circovirus type 2 primer probe are sequentially shown as SEQID No. 25-27.
2. The microfluidic chip according to claim 1, wherein the probe of the primer probe for classical swine fever virus has FAM added to the 5 'end and QSY1 added to the 3' end.
3. The microfluidic chip according to claim 1, wherein FAM is added to the 5 'end of the probe of the porcine pseudorabies virus primer probe, and QSY1 is added to the 3' end of the probe.
4. The microfluidic chip according to claim 1, wherein the probe of the porcine kobuvirus primer probe is added with FAM at the 5 'end and QSY1 at the 3' end.
5. The microfluidic chip according to claim 1, wherein the porcine parvovirus type I primer probe has FAM added to the 5 'end and QSY1 added to the 3' end of the probe.
6. The microfluidic chip according to claim 1, wherein FAM is added to the 5 'end of the probe of the porcine parvovirus type IV primer probe, and QSY1 is added to the 3' end of the probe.
7. The microfluidic chip according to claim 1, wherein FAM is added to the 5 'end of the probe of the swine epidemic encephalitis B virus primer probe, and QSY1 is added to the 3' end of the probe.
8. The microfluidic chip according to claim 1, wherein the probes of the american type porcine reproductive and respiratory syndrome virus primer probes are supplemented with FAM at the 5 'end and QSY1 at the 3' end.
9. The microfluidic chip according to claim 1, wherein the highly pathogenic porcine reproductive and respiratory syndrome virus primer probe has FAM added to the 5 'end and QSY1 added to the 3' end.
10. The microfluidic chip according to claim 1, wherein FAM is added to the 5 'end of the probe of the porcine circovirus type 2 primer probe, and QSY1 is added to the 3' end of the probe.
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| CN202010196399.0A CN111500767A (en) | 2020-03-19 | 2020-03-19 | Microfluid chip for simultaneously detecting 9 porcine pathogens |
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| CN202010196399.0A CN111500767A (en) | 2020-03-19 | 2020-03-19 | Microfluid chip for simultaneously detecting 9 porcine pathogens |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111876475A (en) * | 2020-08-10 | 2020-11-03 | 湖南冠牧生物科技有限公司 | Internal standard composition and application thereof in detection of animal pathogenic microorganisms |
| CN113684309A (en) * | 2021-07-06 | 2021-11-23 | 浙江省动物疫病预防控制中心 | 7 primer probe and kit for detecting viruses related to porcine reproductive disorder diseases based on liquid chip technology and application of primer probe and kit |
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| CN111876475A (en) * | 2020-08-10 | 2020-11-03 | 湖南冠牧生物科技有限公司 | Internal standard composition and application thereof in detection of animal pathogenic microorganisms |
| CN113684309A (en) * | 2021-07-06 | 2021-11-23 | 浙江省动物疫病预防控制中心 | 7 primer probe and kit for detecting viruses related to porcine reproductive disorder diseases based on liquid chip technology and application of primer probe and kit |
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