CN110607393B - Liquid-phase chip detection kit for simultaneously detecting BVDV, IBRV and BPIV, and special primer and coupling probe thereof - Google Patents
Liquid-phase chip detection kit for simultaneously detecting BVDV, IBRV and BPIV, and special primer and coupling probe thereof Download PDFInfo
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Abstract
The invention provides a liquid chip detection kit for simultaneously detecting bovine viral diarrhea-mucosal disease virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus, and a special primer and a coupling probe thereof. The kit disclosed by the invention is simple and convenient to detect BVDV, IBRV, BPIV, has the advantages of strong specificity, high sensitivity and good repeatability, and can be used in the fields of virus detection, vaccine production, calf product quality inspection and the like.
Description
Technical Field
The invention belongs to veterinary animal pathogen detection in the technical field of biological detection, and particularly relates to a liquid chip detection technology capable of simultaneously and specifically detecting Bovine Viral Diarrhea Virus (BVDV), bovine infectious rhinotracheitis virus (IBRV) and bovine parainfluenza virus type 3 (BPIV) and a kit thereof.
Background
Liquid phase chip detection technology
The liquid phase chip detection technology belongs to a high-flux detection technology, and is developed by the American (Luminex) company, integrates the flow cytometry technology, the fluorescent coding microsphere, the laser, the digital signal processing and the traditional biochemical technology, and is a multifunctional analysis platform capable of realizing multi-index simultaneous qualitative and quantitative analysis. The technology has the characteristics of high flux, high sensitivity, good repeatability and sample saving. Since the development of the technology in the middle 90 s of the 20 th century, the application in the field of virus detection has mainly focused on the application of both viral nucleic acid antigens and viral antibodies. The liquid phase chip detection system takes a flow cytometer as a detection platform, takes a plurality of microspheres with uniform and stable sizes as carriers, and the preparation of the microsphere carriers is strictly carried out according to different matching proportions of two classified fluorescent dyes, wherein each dye has 10 matching proportions, the microspheres can be divided into 100 types by mutually combining different proportions, up to 100 signals are detected, and different detection factors are coupled on the microspheres, so that various test purposes can be realized.
Bovine viral diarrhea virus (Bovine Viral Diarrhea virus, BVDV)
Bovine viral diarrhea virus mainly causes bovine viral diarrhea, clinically mainly manifested by fever, mucosal erosion, ulcers, leukopenia, continuous infection, cough, abortion of pregnant cows or abnormal fetuses, and the like, and besides mainly infecting cows, the virus can also cause infection of sheep, pigs, deer and various wild animals, presents worldwide distribution and causes huge economic loss to animal husbandry of various countries. Bovine viral diarrhea viruses can be classified into two biotypes depending on whether they cause epithelial cytopathy, cytopathic and non-cytopathic, usually the non-cytopathic is the majority, and cytopathic is thought to be caused by non-cytopathic genomic mutations, rearrangements, insertions, etc., and cytopathic can be isolated from mucopathic bovine animals. There is no direct link between cytopathic effect and virulence of the strain, but almost all BVDV virulent strains are non-cytogenic, which can cause a great reduction of leukocytes in peripheral blood and is a main cause of acute BVDV infection.
Infectious bovine rhinotracheitis virus (Infectious Bovine Rhinotracheitis virus, IBRV)
Infectious bovine rhinotracheitis virus mainly causes infectious bovine rhinotracheitis, also called necrotic rhinitis of cattle, clinical symptoms are mainly manifested by dyspnea, running water-like nasal discharge and inflammatory reaction of the whole trachea and respiratory mucosa, and can be secondary to other diseases such as meningitis, mammitis and the like of cattle, and can also cause genital tract infection, conjunctival inflammation and systemic infection to cause abortion and stillbirth. The disease is widely spread worldwide, and causes huge economic loss for cattle industry. IBRV has only one serotype, but comprises 3 subtypes, namely IBRV-1.1, IBRV-1.2a, IBRV-1.2b and IBRV-1.3, epidemiology and clinical manifestations of the subtypes are not completely consistent, IBRV-1.1 and IBRV-1.2a mainly cause respiratory tract infection and are represented by infectious rhinotracheitis, IBRV-1.2b can cause infectious pustular vulvovaginitis and the like, and IBRV-1.3 is related to encephalitis and fetal abortion of calves. Subtype 1.2 is weaker in virulence than subtype 1.1 and the transmission capacity of the IBRV-1.1 subtype is strongest.
Bovine parainfluenza virus type 3 (Bovine Parainfluenza virus type, BPIV-3)
Bovine parainfluenza virus type 3 is one of the main pathogens of calf pneumonia, or the main virus causing acute respiratory diseases of cattle and other ruminants, and clinical symptoms are mainly represented by bovine parainfluenza, also called "transport heat", and is an acute contagious disease.
Bovine parainfluenza virus and bovine infectious rhinotracheitis virus, as well as bovine viral diarrhea virus, are the major causative agents of bovine respiratory syndrome (bovine respiratory disease complex, BRDC), and are critical in the prevention of BRDC and in environmental control. In addition, vaccination is the most effective means of preventing diseases as above. In the vaccine production process, in order to prevent the pollution of BVDV, IBRV, BPIV exogenous viruses in bovine raw materials, the accurate, specific and rapid detection of the viruses is performed so as to control the quality, and the method has important significance in the aspects of vaccine production monitoring and the like.
Disclosure of Invention
It is a first object of the present invention to provide a novel high throughput detection technique for simultaneous specific detection of bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus type 3 pathogenic nucleic acid.
The sequences of the labeled primer and the probe for detecting the pathogenic nucleic acid of the bovine viral diarrhea virus provided by the invention are as follows: the nucleotide sequence of the (BVDV-F) upstream primer of BVDV is shown as SED ID NO in the sequence table: 1, the nucleotide sequence of the BVDV downstream primer (BVDV-Biotin-R) is shown as SEQ ID NO:2, the nucleotide sequence of the BVDV coupling probe (BVDV-NH 2-C12-P) is shown as SEQ ID NO: 3.
The sequences of the labeled primer and the probe for detecting the pathogenic nucleic acid of the infectious bovine rhinotracheitis virus provided by the invention are as follows: the nucleotide sequence of the (IBRV-F) upstream primer of the IBRV is shown as SED ID NO:4, the nucleotide sequence of the IBRV downstream primer (IBRV-Biotin-R) is shown as SEQ ID NO:5, the nucleotide sequence of the IBRV coupling probe (IBRV-NH 2-C12-P) is shown as SEQ ID NO: shown at 6.
The sequences of the labeled primer and the probe for detecting bovine parainfluenza virus 3 pathogenic nucleic acid provided by the invention are as follows: the nucleotide sequence of the (BPIV-F) upstream primer of the BPIV is shown as SED ID NO:7, the nucleotide sequence of the BPIV downstream primer (BPIV-Biotin-R) is shown as SEQ ID NO:8, the nucleotide sequence of the BPIV coupling probe (BPIV-NH 2-C12-P) is shown as SEQ ID NO: shown at 9.
Primer/probe sequences derived from the above primers/probes are also included in the present invention. The derivative sequence is shown in SEQ ID NO:1 and/or SEQ ID NO:2 and/or SEQ ID NO:3 and/or SEQ ID NO:4 and/or SEQ ID NO:5 and/or SEQ ID NO:6 and/or SEQ ID NO:7 and/or SEQ ID NO:8 and/or SEQ ID NO:9 by substitution, deletion or addition of one to ten bases.
It is a second object of the present invention to provide a kit for simultaneous liquid phase chip detection of BVDV, IBRV and BPIV.
The liquid chip detection kit provided by the invention comprises the primer and the labeled probe for liquid chip detection of BVDV, IBRV and BPIV.
Specifically, the kit comprises:
(1) Reagents for a 25. Mu.L conventional PCR reaction system: one-Step PCR reaction solution 2 XOne Step RT-PCR Buffer III 12.5. Mu.L (from TakaRa Co., ltd.), taKaRa Ex Taq HS 0.5. Mu.L (from TakaRa Co., ltd.), primeScript RT Enzyme Mix II 0.5.5. Mu.L (from TakaRa Co., ltd.), BVDV-F/IBRV-F/BPIV-F (20. Mu.M) each 0.25. Mu.L, BVDV-Biotin-R/IBRV-Biotin-R/BPIV-Biotin-R (20. Mu.M) each 0.5. Mu.L, RNA-free H 2 O7.25. Mu.L, nucleic acid template 2. Mu.L.
(2) Magnetic beads, 44/54/14 (available from luminex corporation), SAPE chromogenic solution (available from invitrogen corporation) and 1×tmac and 1.5×tmac buffers, respectively, to which the corresponding conjugate probes have been conjugated, are used for BVDV/IBRV/BPIV virus detection.
For convenient detection, the kit also comprises a positive control and a negative control, wherein the positive control is bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus genome nucleic acid, and the negative control is a reaction system which does not contain bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus, such as H 2 O (double distilled water, sterile deionized water, etc.).
The kit may further comprise instructions describing the procedure.
It is a third object of the present invention to provide the detection of the primers, probes or kits for the purpose of non-disease diagnosis of Bovine Viral Diarrhea Virus (BVDV), bovine infectious rhinotracheitis virus (IBRV) and bovine parainfluenza virus type 3 (BPIV-3).
One of the applications is a qualitative detection method for non-disease diagnosis purpose of Bovine Viral Diarrhea Virus (BVDV), bovine infectious rhinotracheitis virus (IBRV) and bovine parainfluenza virus type 3 (BPIV-3) by using the liquid phase chip detection technology, which comprises the following steps:
1) Coupling the coupling probes of each virus with the corresponding magnetic balls of each virus;
2) Extracting genome RNA/DNA of a sample to be detected, and carrying out ordinary PCR amplification detection under the guidance of the primers by taking the extracted genome RNA/DNA as a template;
3) The PCR amplified products are hybridized with the magnetic balls coupled with the virus probes, and liquid phase chip analysis is carried out, so that qualitative detection of bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus is realized.
Specifically, the liquid phase chip detection method comprises the following steps:
1) Coupling the coupling probes of the viruses with the corresponding magnetic spheres of the viruses, wherein the coupling probes for detecting Bovine Viral Diarrhea Virus (BVDV)/bovine infectious rhinotracheitis virus (IBRV)/bovine parainfluenza virus (BPIV) are respectively coupled with the magnetic spheres with the numbers of 44/54/14;
2) Extracting genome RNA/DNA of a sample to be detected, and carrying out ordinary PCR amplification detection under the guidance of the primers by taking the extracted genome RNA/DNA as a template;
3) Coupling magnetic spheres with the obtained PCR amplification products and each virus probe, developing by using SAPE, and analyzing a liquid phase chip to realize qualitative detection of Bovine Viral Diarrhea Virus (BVDV)/bovine infectious rhinotracheitis virus (IBRV)/bovine parainfluenza virus (BPIV);
4) Result criterion (Qualitative criteria, QC):
A. when the Positive and negative MFI ratio (PN) is more than or equal to 3 and the Positive MFI is more than or equal to 300, the test is established, QC is equal to the ratio of an MFI sample to a negative control (N-control), namely QC=MFI sample/N-control,
B.A when the test is established, judging that the MFI sample of each virus magnetic sphere is more than or equal to 300, QC is more than or equal to 3, and judging that the sample is a corresponding virus positive sample; otherwise, judging as negative; namely:
if the MFI value of the No. 44 magnetic sphere is lower than 300, the sample is free of bovine viral diarrhea virus, and if the MFI value of the No. 44 magnetic sphere is more than or equal to 300 and QC is more than or equal to 3, the sample is free of bovine viral diarrhea virus; if the MFI value of the 54 # magnetic ball is lower than 300, the sample is free of infectious bovine rhinotracheitis virus, and if the MFI value of the 54 # magnetic ball is more than or equal to 300 and QC is more than or equal to 3, the sample is free of infectious bovine rhinotracheitis virus; if the MFI value of the No. 14 magnetic sphere is lower than 300, the sample is free of bovine parainfluenza virus, and if the MFI value of the No. 14 magnetic sphere is more than or equal to 300 and QC is more than or equal to 3, the sample is free of bovine parainfluenza virus;
in the above method, the sample to be tested in step 2) may be serum taken from calves for vaccine production, or may be primary cells isolated from the testes of calves, which are detected for non-diagnostic purposes. The qualitative detection of BVDV, IBRV and BPIV viruses in the sample to be detected is used for monitoring calf-based products and raw materials and providing objective data for subsequent raw material treatment.
The general PCR reaction system in the step 2) can comprise: one-Step PCR reaction solution 2 XOne Step RT-PCR Buffer III 12.5. Mu.L (purchased from TakaRa Co., ltd.), taKaRa Ex Taq HS 0.5. Mu.L (purchased from TakaRa Co., ltd.), primeScript RT Enzyme Mix II 0.5.5. Mu.L (purchased from TakaRa Co., ltd.), BVDV-F/IBRV-F/BPIV-F (20. Mu.M) 0.25. Mu.L each, BVDV-Biotin-R/IBRV-Biotin-R/BPIV-Biotin-R (20. Mu.M) 0.5. Mu.L each, RNA-free H2O 7.25. Mu.L, nucleic acid template 2. Mu.L, primer dilution 20 ng/. Mu.L, and final addition of 10ng and 5ng in the reaction system.
The general PCR reaction conditions in the step 2) can be as follows: firstly, the temperature is 52 ℃ for 15min and 95 ℃ for 2min; then 94 ℃ for 30s,56 ℃ for 30s,72 ℃ for 30s,35 cycles; finally, the final extension is carried out at 72 ℃ for 7min.
The invention provides a novel high-flux detection technology, which can realize the specific detection of various virus nucleic acids by using magnetic balls with different numbers and colors and coupling probes. The invention can realize the rapid detection of bovine viral diarrhea virus/bovine infectious rhinotracheitis virus/bovine parainfluenza virus, can provide a rapid and effective technical means for screening exogenous viruses of bovine raw materials, ensures the safety and purity of vaccination, can monitor the quality of calf raw materials, and provides objective data for subsequent treatment so as to ensure the safety of calf serum, cells and products thereof. The kit and the detection method have the advantages of simple and convenient operation, strong specificity, high sensitivity, good repeatability and high flux, and can play an important role in vaccine production and calf product production (application for non-diagnosis purpose), and have wide application prospect.
The present invention will be described in further detail with reference to specific examples.
Drawings
FIG. 1 is a schematic diagram of a qualitative detection technique for bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, bovine parainfluenza virus using liquid phase chip detection technique according to the present invention.
FIG. 2 shows the result of detecting the coupling efficiency of the coupling probe of the No. 44 magnetic ball and the bovine viral diarrhea virus.
FIG. 3 shows the result of detecting the coupling efficiency of the 54 # magnetic ball and the coupling probe of the infectious bovine rhinotracheitis virus.
FIG. 4 shows the result of detecting the coupling efficiency of the coupling probe of the No. 14 magnetic ball and bovine parainfluenza virus.
Detailed Description
The methods used in the examples below are conventional methods unless otherwise specified, and specific steps can be found in: molecular Cloning: A Laboratory Manual (Sambrook, J., russell, david W., molecular Cloning: A Laboratory Manual,3rd edition,2001,NY,Cold Spring Harbor).
The percentage concentrations are mass/mass (W/W, unit g/100 g) percentage concentration, mass/volume (W/V, unit g/100 mL) percentage concentration or volume/volume (V/V, unit mL/100 mL) percentage concentration unless otherwise specified.
The various biomaterials described in the examples were obtained by merely providing an experimental route for achieving the objectives of the specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples.
The labeled primer and the coupled probe were synthesized by TaKaRa gene Co.
Examples detailed embodiments and specific operation procedures are given on the premise of the technical scheme of the present invention, and examples will help to understand the present invention, but the protection scope of the present invention is not limited to the following examples.
The biological genome is one of the most objective indexes for directly reflecting basic biological information, different viruses contain different genome information, different viruses can be classified into different groups through the genome information, and a large amount of amplification of specific locus gene sequences can be realized by utilizing the principle of genome base complementation pairing, so that the viruses are visible to naked eyes through a certain method.
As shown in FIG. 1, the invention designs 3 pairs of specific labeled primers and 3 coupled probes based on the basic principle, and establishes a liquid phase chip detection method for simultaneously and specifically detecting BVDV/IBRV/BPIV by utilizing the basic complementary pairing principle.
Example 1 design of labeled primers and coupled probes for detection of BVDV/IBRV/BPIV Virus Using liquid phase chip technology
BVDV detection sequence selection: the conserved 5' UTR gene sequences (SED ID NO:10 and SED ID NO: 11 in the sequence table) of BVDV type I virus and BVDV type II virus are respectively selected, BVDV type I virus and BVDV type II virus can be amplified simultaneously, the design of coupling probes and labeled primers is carried out according to the design principle of liquid phase labeled primers and coupling probes, multiple groups of primers are obtained, and the optimal primer sequence information finally determined based on experimental results is as follows:
BVDV-F (upstream primer): 5'-AGCAACAGTGGTGAGTTCGTTGGA-3' (SED ID NO:1 in the sequence Listing).
BVDV-R (downstream primer): 5'-biotin-GCCCTCGTCCACGTGGYATCTCG-3' (SED ID NO:2 in the sequence Listing).
BVDV-P (conjugate probe): 5 '-amino-linker C12 (12 carbons) -AGTACAGGGTAGTCGTCARTGGTTCG-3' (SED ID NO:3 in the sequence Listing).
Selecting an IBRV detection sequence: the IBRV virus selects the conserved gE gene sequence (SED ID NO: 12) because the IBRV vaccine strain always lacks the gE gene, but the wild strain does not. The method can realize the detection of wild strains without detecting vaccine strains, and the design of the coupling probes and the labeling primers is carried out according to the design principle of the liquid-phase labeling primers and the coupling probes, so that a plurality of groups of primers are obtained, and the optimal primer sequence information finally determined based on experimental results is as follows:
IBRV-F (upstream primer): 5'-AGTTTGGTTTAGGGACCCGCTT-3' (SED ID NO:4 in the sequence Listing).
IBRV-R (downstream primer): 5'-biotin-CGCTGCTACCACGGTGTAATCT-3' (SED ID NO:5 in the sequence Listing).
IBRV-P (coupled probe): 5 '-amino-linker C12 (12 carbons) -CGATGCCGCGCCAGCGCGGACCC-3' (SED ID NO:6 in the sequence Listing).
Selection of a BPIV detection sequence: the BPIV virus selects a conserved P gene sequence (SED ID NO: 13) thereof, and designs a coupling probe and a labeling primer according to the design principle of a liquid-phase labeling primer and the coupling probe to obtain a plurality of groups of primers, and the optimal primer sequence information finally determined based on experimental results is as follows:
BPIV-F (upstream primer): 5'-GCGCGATGACATCATTACAG-3' (SED ID NO:7 in the sequence Listing).
BPIV-R (downstream primer): 5'-biotin-ACAACTCTCTTGTCTTGGTC-3' (SED ID NO:8 in the sequence Listing).
BPIV-P (conjugate probe): 5 '-amino-linker C12 (12 carbons) -TCTDGGTGTCATCCAAGCTGCAGCA-3' (SED ID NO:9 in the sequence Listing).
Example 2 verification of coupling efficiency Using the coupling probes of the invention and the luminex magnetic spheres
1. Coupling of coupling probes with luminex magnetic spheres
And respectively selecting a 44/54/14 number luminex magnetic sphere to couple with a corresponding BVDV/IBRV/BPIV coupling probe, wherein the coupling flow is as follows:
(1) Diluting the coupling probe, and diluting the synthesized coupling probe with the 5' -end marked amino to 100 mu moL;
(2) The luminex magnetic pellet was returned to room temperature and mixed upside down and 100. Mu.L (1.25X10) 6 ) Centrifuging at 12000rpm in a 1.5mL sterile and enzyme-free centrifuge tube for 2min, and removing supernatant;
(3) 50. Mu.L of 0.1M MES pH4.5 solution stored at 4 ℃ is added and blown and mixed uniformly;
(4) Adding 5 mu L of 0.1mM coupling probe, and sucking and beating uniformly;
(5) Preparing EDC solution, taking about 10mg of EDC dry powder, dissolving the EDC dry powder in 1.5mL of sterile centrifuge tube with 1mL of purified water, adding 5 mu L of the EDC dry powder into the 4-step solution, blowing and mixing uniformly, and incubating at room temperature in a dark place for 30min;
(6) Repeating the step 5;
(7) 1mL of 0.02% Tween-20 solution is added into a 1.5mL sterile and aseptic centrifuge tube, and the mixture is blown and mixed uniformly, centrifuged at 12000rpm for 2min, and the supernatant is sucked and removed;
(8) 1mL of 0.1% SDS solution is added into a 1.5mL sterile and aseptic centrifuge tube, and the mixture is blown and evenly mixed, centrifuged at 12000rpm for 2min, and the supernatant is sucked and removed;
(9) 100 mu L of TE pH8.0 solution is added into a 1.5mL sterile and aseptic centrifuge tube, and the mixture is blown and evenly mixed, and the mixture is stored at 2-8 ℃ after being counted by a cell counter in a dark place.
2. Coupling efficiency validation
(1) Diluting the coupled magnetic balls to 100 pieces/mu L by using 1.5 xTMAC according to the counting result of the coupled magnetic balls;
(2) Diluting the coupling verification biotin-labeled primer (i.e., the reverse complement of the 5' -end biotin-labeled coupling probe) to 10 fmol/. Mu.L;
(3) Preparing a verification system, adding 33 mu L of magnetic spheres and a coupling verification biotin-labeled primer into a 60 mu L system, wherein the concentration of the magnetic spheres and the coupling verification biotin-labeled primer is sequentially 0fmol, 25fmol, 50fmol, 100fmol, 125fmol, 150fmol, 175fmol, 200fmol and 225fmol, and supplementing the mixture to 60 mu L by using water;
(4) Denaturation at 96℃for 5min, hybridization at 50℃for 15min;
(5) The SAPE was diluted to 10mg/ml with 1 XPAC;
(6) 25. Mu.L of SAPE was added to the hybridization system, and the mixture was stirred and mixed well, incubated at 50℃for 5 minutes, and assayed by a luminex 200 instrument at 50 ℃.
The coupling efficiency results are shown in FIGS. 2-4: along with the sequential increment of the concentration of the biotin-labeled primer in the coupling verification, the MFI value detected by the liquid phase chip is sequentially incremented, and can be higher than 10000MFI at maximum, so that the coupling is proved to be successful, and the coupling efficiency is high.
Example 3 qualitative detection of Bovine Viral Diarrhea Virus (BVDV), bovine infectious rhinotracheitis Virus (IBRV) and bovine parainfluenza Virus type 3 (BPIV-3) by the method of the invention
1. Extracting genomic nucleic acid of a sample to be tested
Cell cultures (positive control) of inactivated bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus, respectively, and test samplesThe method for extracting genomic RNA/DNA comprises the following steps (Axyprep TM Body Fluid Viral DNA/RNA Miniprep Kit, AXYGEN):
(1) AXYGEN kit preparation: isopropyl alcohol containing 1% glacial acetic acid and absolute ethanol of a specified concentration were prepared in advance according to the kit instructions and added to the reagents Buffer W1A and Buffer W2.
(2) 200 mu L of sample to be detected is added into a 1.5mL centrifuge tube, 200 mu L of Buffer V-L is added, and after vortex shaking and mixing, the mixture is kept stand for 5min.
(3) Add 75. Mu.L Buffer V-N to 1.5mL sample and reagent mixing centrifuge tube of step (2), mix well with vortex shaking, centrifuge 12000g for 5min.
(4) The supernatant was transferred to a 2mL centrifuge tube (provided in the kit), 300. Mu.L of isopropyl alcohol (1% glacial acetic acid) was added, and the mixture was inverted 6-8 times and mixed well.
(5) The preparation tube was placed in a 2mL centrifuge tube (provided in the kit), and the mixed solution in step (4) was transferred into the preparation tube, and centrifuged at 6000g for 1min.
(6) The filtrate was discarded, the preparation tube was returned to a 2mL centrifuge tube, 500. Mu.L Buffer W1A was added, and the mixture was allowed to stand at room temperature for 1min. Centrifuge 12000g for 1min.
(7) The filtrate was discarded, the preparation tube was placed back into a 2mL centrifuge tube, 800. Mu.L Buffer W2 was added, and 12000g was centrifuged for 1min.
(8) The preparation tube was placed back into a 2mL centrifuge tube and centrifuged at 12000g for 1min.
(9) The preparation tube was placed in a clean 1.5mL centrifuge tube (provided in the kit), 40. Mu.L of enzyme-free water was added to the center of the preparation tube membrane, and the tube was left to stand at room temperature for 1min, and 12000g was centrifuged for 1min to elute RNA/DNA.
2. Common PCR amplification and liquid chip detection of Bovine Viral Diarrhea Virus (BVDV), bovine infectious rhinotracheitis virus (IBRV) and bovine parainfluenza virus type 3 (BPIV-3) in sample to be detected
1. PCR amplification of test sample, negative control and positive control
The genomic RNA/DNA from Bovine Viral Diarrhea Virus (BVDV), bovine infectious rhinotracheitis virus (IBRV) and bovine parainfluenza virus type 3 (BPIV-3), negative control and test sample RNA/DNA extracted in the step one were subjected to PCR amplification, and a 25. Mu.L reaction system is shown in Table 1.
TABLE 1 BVDV, IBRV and BPIV common PCR amplification System
| Reagent name | Volume (mu L) |
| 2 XOne Step RT-PCR Buffer III (from TaKaRa Co.) | 12.5 |
| TaKaRa Ex Taq HS (from TakaRa Co.) | 0.5 |
| PrimeScript RT Enzyme Mix II (from TakaRa company) | 0.5 |
| BVDV-F/IBRV-F/BPIV-F (20. Mu.M) each: | 0.25 |
| BVDV-R/IBRV-R/BPIV-R (20. Mu.M) each: | 0.5 |
| RNA template | 2.0 |
| RNA-free H 2 O | 7.25 |
The PCR reaction cycle conditions are shown in Table 2.
TABLE 2 circulation conditions for BVDV/IBRV/BPIV ordinary PCR
2. Liquid chip detection of sample to be detected, negative control and positive control
Coupling magnetic balls and SAPE color developing solution are diluted 100 times respectively, wherein the coupling magnetic balls are diluted by 1.5 xTMAC buffer solution, the SAPE color developing solution is diluted by 1 xTMAC buffer solution, and a liquid phase chip detection system is configured: 33 mu L magnetic ball and 12 mu L H 2 0 and 5. Mu.L of PCR amplification product, pre-denaturing at 96℃for 5min, hybridizing at 50℃for 15min, adding 25. Mu.L of SAPE color development solution, mixing well, standing at 50℃for 5min, and performing on-machine analysis at 50 ℃.
3. Result criterion (Qualitative criteria, QC)
When the Positive and negative MFI ratio (PN) is more than or equal to 3 and the Positive MFI is more than or equal to 300, the test is established, and QC is equal to the ratio of an MFI sample to a negative control (N-control), namely QC=MFI sample/N-control.
When the test is judged to be established, judging that the MFI sample of each virus magnetic sphere is more than or equal to 300, QC is more than or equal to 3, and judging that the sample is a corresponding virus positive sample; otherwise, the method is judged as negative, namely: if the MFI value of the No. 44 magnetic sphere is lower than 300, the sample is free of bovine viral diarrhea virus, and if the MFI value of the No. 44 magnetic sphere is more than or equal to 300 and QC is more than or equal to 3, the sample is free of bovine viral diarrhea virus; if the MFI value of the 54 # magnetic ball is lower than 300, the sample is free of infectious bovine rhinotracheitis virus, and if the MFI value of the 54 # magnetic ball is more than or equal to 300 and QC is more than or equal to 3, the sample is free of infectious bovine rhinotracheitis virus; if the MFI value of the No. 14 magnetic sphere is lower than 300, the sample is free of bovine parainfluenza virus, and if the MFI value of the No. 14 magnetic sphere is more than or equal to 300 and QC is more than or equal to 3, the sample is free of bovine parainfluenza virus;
example 4 specificity experiments of the detection method of the invention
Genomic RNA/DNA was extracted from Bovine Epidemic Fever Virus (BEFV), bovine syncytial virus (BRSV), foot and Mouth Disease Virus (FMDV), pasteurella (PM), brucella (bru) using the DNA/RNA simultaneous extraction AxyGen kit (available from Corning Life sciences (Wu Jiang)) as described in example 3, positive controls were made for inactivated Bovine Viral Diarrhea Virus (BVDV), bovine infectious rhinotracheitis virus (IBRV), bovine parainfluenza virus (BPIV), negative controls were made for non-enzymatic water, and liquid chip assays were performed under the guidance of the primers, conjugate probes of the invention, and the liquid chip assay method was referred to example 3 to verify the specificity of the method.
The detection results are shown in Table 3, the MFI values of specific nucleic acids BEFV, BRSV, FMDV, PM and bru are lower than 300, and corresponding positive results appear in different positive control samples, so that the detection results show that bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus can be specifically detected by the method.
TABLE 3 specificity detection results of the liquid chip detection methods for bovine viral diarrhea Virus, bovine infectious rhinotracheitis Virus and bovine parainfluenza Virus
Example 5 sensitivity test of the detection method of the present invention
Will 10 8 Positive plasmid nucleic acid samples of the bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus of the copies/. Mu.L are diluted sequentially according to a 10-fold gradient to 10 concentrations respectively 1 The detection of the liquid phase chip method was carried out under the guidance of the primers and the coupled probes of the present invention using nucleic acid samples of different concentrations as templates, and the liquid phase chip test method was referred to example 3 to verify the sensitivity of the method.
The results are shown in Table 4, 10 8 To 10 1 Respectively represent BVDV&IBRV&8 different concentration gradients of BPIV, wherein the MFI values of the 8 concentration gradients are all more than 300, and the QC values are all more than 3, and the result shows that the detection sensitivity of the triple liquid-phase chip detection method for bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus is 10 1 copies/μL。
TABLE 4 sensitivity test results of liquid-phase chip detection methods for bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus
Example 6 repeatability experiments of the detection method of the invention
A total of 8 gradient nucleic acid samples, 10-fold gradient diluted as described in example 5, were each subjected to 3 replicates, and subjected to a liquid phase chip assay under the direction of the primers and conjugate probes of the invention, with reference to example 3, to verify the reproducibility of the assay.
The detection results are shown in Table 5, and the 3 repeated samples of each concentration gradient are visible from the table, and the detection results are consistent, so that the repeatability of the triple liquid phase chip detection method of the bovine viral diarrhea virus, the bovine infectious rhinotracheitis virus and the bovine parainfluenza virus is better.
TABLE 5 repeated detection results of bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus liquid phase chip detection method
Example 7 liquid phase chip detection kit for detection of Bovine Viral Diarrhea Virus (BVDV), bovine infectious rhinotracheitis Virus (IBRV) and bovine parainfluenza Virus (BPIV)
Based on examples 1, 2 and 3, the liquid phase chip detection kit provided by the invention comprises primers and coupled magnetic balls (with probes) for liquid phase chip detection of bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus.
Specifically, the kit comprises the following reagents for a 25. Mu.L common PCR reaction system: one-Step PCR reaction solution 2 XOne Step RT-PCR Buffer III 12.5. Mu.L (purchased from TakaRa Co., ltd.), taKaRa Ex Taq HS 0.5. Mu.L (purchased from TakaRa Co., ltd.), primeScript RT Enzyme Mix II 0.5.5. Mu.L (purchased from TakaRa Co., ltd.), BVDV-F/IBRV-F/BPIV-F (20. Mu.M) 0.25. Mu.L each, BVDV-Biotin-R/IBRV-Biotin-R/BPIV-Biotin-R/(20. Mu.M) 0.5. Mu.L each, RNA-free H2O 7.25. Mu.L, nucleic acid template 2. Mu.L, primer dilution 20 ng/. Mu.L, and final addition of 10ng and 5ng in the reaction system.
The kit also comprises SAPE chromogenic solution, 1 xTMAC buffer solution and 1.5 xTMAC buffer solution, and can also comprise positive control and negative control for detection, wherein the positive control is bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus genome RNA/DNA, and the negative control is a reaction system free of bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus genome RNA/DNA, such as H 2 O (double distilled water, sterile deionized water, etc.).
The use of the reagents in the kit can be referred to in example 3.
Example 8 qualitative detection of Bovine Viral Diarrhea Virus (BVDV), bovine infectious rhinotracheitis Virus (IBRV) and bovine parainfluenza Virus (BPIV) genomic RNA/DNA exogenous Virus by raw materials
The method for detecting BVDV, IBRV, BPIV exogenous virus in bovine-derived raw material in vaccine production was the same as in example 3. The sample to be tested in this embodiment is bovine serum.
The test results are shown in Table 6, and it can be seen from the table that in 16 parts of bovine serum, 4 parts of exogenous virus BVDV pollutes, 2 parts of exogenous virus IBRV pollutes, 1 part of exogenous virus BPIV pollutes, 1 part of mixed infection of BVDV and IBRV, 1 part of mixed infection of BVDV and BPIV, and 11 parts of exogenous virus BVDV, IBRV and BPIV pollutes.
TABLE 6 detection results of liquid chip detection methods of bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus in samples of different composition
Sequence listing
<110> Jin Yubao Programmes Biochemical Co., ltd
<120> liquid chip detection kit for simultaneously detecting BVDV, IBRV and BPIV, and special primer and coupling probe thereof
<130> CGCNB185056W
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
agcaacagtg gtgagttcgt tgga 24
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gccctcgtcc acgtggyatc tcg 23
<210> 3
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
agtacagggt agtcgtcart ggttcg 26
<210> 4
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
agtttggttt agggacccgc tt 22
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
cgctgctacc acggtgtaat ct 22
<210> 6
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
cgatgccgcg ccagcgcgga ccc 23
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
gcgcgatgac atcattacag 20
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
acaactctct tgtcttggtc 20
<210> 9
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
tctdggtgtc atccaagctg cagca 25
<210> 10
<211> 600
<212> DNA
<213> BVDV type I virus 5' UTR Gene sequence (Bovine Viral Diarrhea virus)
<400> 10
gtatacgaga atttgcctaa cctcgtatac acattgggca ctctaaaaat gaattaggcc 60
tgagggacaa atcctcctta gcgaaggccg aaaagaggct agccatgccc ttagtaggac 120
tagcaaaaca aggagggtag caacagtggt gagttcgttg gatggctgaa gccctgagta 180
cagggtagtc gtcagtggtt cgacgctttg tgcgacaagc ctcgagatgc cacgtggacg 240
agggcatgcc cacagcacat cttaacctga gcgggggtcc ttcaggtgga aacggtgtaa 300
ccaaccgcta cgaatacagc ctgatagggt gctgcagagg cccactgtat cgctactaaa 360
actatctgct gtacatggca catggagttg atcacaaatg aacttttata caaaacatac 420
aaacaaaaac ccgctggagt ggaggaacca gtgtacaacc gagcgggcga ccctttgttt 480
ggcgagagag gagaggttca tccgcaggca acgctaaaac taccacataa aagaggggag 540
cgtgaagttc ctactaattt ggcgtctcta ccaaaaagag gtgactgcag atcgggtaac 600
<210> 11
<211> 257
<212> DNA
<213> BVDV type II virus 5' UTR Gene sequence (Bovine Viral Diarrhea virus)
<400> 11
catgccctta gtaggactag caaaaggagg ggactagcgg tagcagtgag ttcattggat 60
ggccgaatcc ctgagtacag ggaagtcgtc aatggttcga cactccatca atcgaggagt 120
ctcgagatgc catgtggacg agggcatgcc cacggcacat cttaacctat gcgggggttg 180
catgggtgaa agcaccattc gtggtgttat ggacacagcc tgatagggtg tagcagagac 240
ctgctattcc gctagta 257
<210> 12
<211> 1793
<212> DNA
<213> IBRV virus gE Gene sequence (Infectious Bovine Rhinotracheitis virus)
<400> 12
atgcaaccca ccgcgccgcc ccggcggcgg ttgctgccgc tgctgctgcc gcagttattg 60
cttttcgggc tgatggccga ggccaagccc gcgaccgaaa ccccgggctc ggcttcggtc 120
gacacggtct tcacggcgcg cgctggcgcg cccgtctttc tcccagggcc cgcggcgcgc 180
ccggacgtgc gcgccgttcg cggctggagc gtcctcgcgg gcgcctgctc gccgcccgtg 240
ccggagcccg tctgcctcga cgaccgcgag tgcttcaccg acgtggccct ggacgcggcc 300
tgcctgcgaa ccgcccgcgt ggccccgctg gccatcgcgg agctcgccga gcggcccgac 360
tcaacgggcg acaaagagtt tgttctcgcc gacccgcacg tctcggcgca gctgggtcgc 420
aacgcgaccg gggtgctgat cgcggccgca gccgaggagg acggcggcgt gtacttcctg 480
tacgaccggc tcatcggcga cgccggcgac gaggagacgc agttggcgct gacgctgcag 540
gtcgcgacgg ccggcgcgca gggcgccgcg cgggacgagg agagggaacc agcgaccggg 600
cccacccccg gcccgccgcc ccaccgcacg acgacacgcg cgcccccgcg gcggcacggc 660
gcgcgcttcc gcgtgctgcc gtaccactcc cacgtataca ccccgggcga ttcctttctg 720
ctatcggtgc gtctgcagtc tgagtttttc gacgaggctc ccttctcggc cagcatcgac 780
tggtacttcc tgcggacggc cggcgactgc gcgctcatcc gcatatacga gacgtgcatc 840
ttccaccccg aggcaccggc ctgcctgcac cccgccgacg cgcagtgcag cttcgcgtcg 900
ccgtaccgct ccgagaccgt gtacagccgg ctgtacgagc agtgccgccc ggaccctgcc 960
ggtcgctggc cgcacgagtg cgagggcgcc gcgtacgcgg cgcccgttgc gcacctgcgt 1020
cccgccaata acagcgtaga cctggtcttt gacgacgcgc cggctgcggc ctccgggctt 1080
tacgtctttg tgctgcagta caacggccac gtggaagctt gggactacag cctagtcgtt 1140
acttcggacc gtttggtgcg cgcggtcacc gaccacacgc gccccgaggc cgcagccgcc 1200
gacgctcccg agccaggccc accgctcacc agcgagccgg cgggcgcgcc caccgggccc 1260
gcgccctggc ttgtggtgct ggtgggcgcg cttggactcg cgggactggt gggcatcgca 1320
gccctcgccg ttcgggtgtg cgcgcgccgc gcaagccaga agcgcaccta cgacatcctc 1380
aaccccttcg ggcccgtata caccagcttg ccgaccaacg agccgctcga cgtggtggtg 1440
ccagttagcg acgacgaatt ttccctcgac gaagactctt ttgcggatga cgacagcgac 1500
gatgacgggc ccgctagcaa cccccctgcg gatgcctacg acctcgccgg cgccccagag 1560
ccaactagcg ggtttgcgcg agcccccgcc aacggcacgc gctcgagtcg ctctgggttc 1620
aaagtttggt ttagggaccc gcttgaagac gatgccgcgc cagcgcggac cccggccgca 1680
ccagattaca ccgtggtagc agcgcgactc aagtccatcc tccgctaggc gccccccccc 1740
ccgcgcgctg tgccgtctga cggaaagcac ccgcgtgtag ggctgcatat aaa 1793
<210> 13
<211> 1803
<212> DNA
<213> BPIV Virus P Gene sequence (Bovine Parainfluenza virus type 3)
<400> 13
atggaagaca atgttcaaaa caatcaaatc atggattctt gggaagagag atcaggagac 60
aagtcatccg acatctcatc agccctcgac atcattgaat tcatactcag caccgatccc 120
caagaaagcg cggccagcgt caatgagaac ggcgcagagg tcacaaggct cagcacaaca 180
atccaccggc ccgacccaaa atcaacaaag acaagcgagg aaaatagtgg atcaactaac 240
gaagatcgac agcttgggac atcatacaaa tgtaccgcag aaacaaaaga tagagttgtt 300
aatcaggaag ctatacaggg aagaaatagg agaggaagca gctcagatag tagaactgag 360
actatggtca ttagaagaat caccagaggc agctcagatc ctgacaatgg aaccgaaatc 420
caggaaaatc ttgattacaa tgaagctaga gaggtggata agaaccctac taaggggaaa 480
gtgcgacaac ttgaaaatgt tccagtcaag gtaccaagaa gtgatgccat acctccagca 540
aaatcagatg gaaacagtga tgatggagga agcctggaat ctatcagtac acctaatcca 600
agacatacta gcctagtgac cacagcaaca ccagatgatg aagaggagct tctctcgaaa 660
aacaaaaggt caaaaaggca ccagctaaca aaccagagag acaacaaaga aattaaaaaa 720
gggggagagg aggatatttg gaagcaagca gatatcgacc atcagtcatc cagattggac 780
tacgagcccg acttcaaagg atcgaagagg aaccagagga ccctcaagac cagcacagat 840
gcagaggaac caacaagaat acagaatggg tcccaaagga agagaatcac atcctggaac 900
atcctcaaca acaagagcag caatcgagag gaacagacag tcagaaatcc ccaacgatca 960
gcacacggac agaagcaaac aatggtatca gacagatcag cccccgaaca acaagtcaaa 1020
gcacagaaga cgaacggaga ggaaagaaag gacacagaag agagcactca atttacagaa 1080
agggcgatta cattattaca gagtcttggt gtaatccaat ctgcagcaaa attagacctg 1140
taccaagaca agagagttgt atgtgtggcg aatgtcctaa acaatgcaga tactgcatcg 1200
aagatcgatt ttttagcggg tctgatgatt ggagtatcga tggatcagga caacagactt 1260
aatcagattc aaaatgagat aatagattta aagactgatc tcaaaaagat ggatgaatca 1320
cacagaaggt tgattgagaa tcaaaaagaa caactatcat taatcacatc attaatttcg 1380
aatcttaaga ttatgacaga gagaggagga aagaaagatc aggttgagac aagtggaaga 1440
acaccaatga ttaaaacaaa aatgaaagaa gaaaagataa ggaaagttaa gttcgatcct 1500
ctcatggaaa cacaaggcat cgaaaagaat gttcctgatt tatacaggtc gatagagaag 1560
gcaccagaaa gtgacgtgca gatcaaatca gaaatcagta ggttaagtga tgaatcgaat 1620
gcaactagac taataccaaa gagaataagc agcacaatga ggtcattaat cataatcatc 1680
aataacagca gcctgtcatc aaaggcaaaa cagtcataca taaatgaact caagctctgc 1740
aagagtgatg aagaagtatc tgaattgatg gatatgttta atgaagacat tagctcccaa 1800
tag 1803
Claims (6)
1. An oligonucleotide composition for use in a liquid phase chip consisting of a primer pair and a coupling probe for detecting Bovine Viral Diarrhea Virus (BVDV), a primer pair and a coupling probe for detecting bovine infectious rhinotracheitis virus (IBRV), and a primer pair and a coupling probe for detecting bovine parainfluenza virus (BPIV), wherein the BVDV upstream primer is: 5'-AGCAACAGTGGTGAGTTCGTTGGA-3', BVDV downstream primers are: the 5'-biotin-GCCCTCGTCCACGTGGYATCTCG-3', BVDV coupling probes are: 5 '-amino-linker C12-AGTACAGGGTAGTCGTCARTGGTTCG-3'; the IBRV upstream primer is: 5'-AGTTTGGTTTAGGGACCCGCTT-3', IBRV downstream primers are: the 5'-biotin-CGCTGCTACCACGGTGTAATCT-3' IBRV coupled probe comprises the following components: 5 '-amino-linker C12-CGATGCCGCGCCAGCGCGGACCC-3'; the BPIV upstream primer is: 5'-GCGCGATGACATCATTACAG-3', BPIV downstream primers are: the 5'-biotin-ACAACTCTCTTGTCTTGGTC-3' BPIV coupling probe is: 5 '-amino-linker C12-TCTDGGTGTCATCCAAGCTGCAGCA-3'.
2. A kit for liquid phase chip detection of bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, and bovine parainfluenza virus comprising the oligonucleotide composition of claim 1.
3. The kit of claim 2, wherein: the kit comprises the following 25 [ mu ] L PCR reaction system: one-Step PCR reaction solution 2 XOne Step RT-PCR Buffer III 12.5. Mu.L, taKaRa Ex Taq HS 0.5. Mu.L, primeScript RT Enzyme Mix II 0.5.5. Mu.L, BVDV upstream primer/IBRV upstream primer/BPIV upstream primer each 0.25. Mu.L, BVDV downstream primer/IBRV downstream primer/BPIV downstream primer each 0.5. Mu.L, RNA-free H 2 O7.25. Mu.L, nucleic acid template 2. Mu.L, all primer concentrations were 20 ng/. Mu.L.
4. A kit according to claim 2 or 3, wherein: the kit also comprises a positive control and a negative control, wherein the positive control is bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus nucleic acid, and the negative control is a reaction system which does not contain bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus.
5. A method of detecting bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, and bovine parainfluenza virus with the kit of any one of claims 2-4 for non-disease diagnostic purposes, comprising the steps of:
1) Coupling the BVDV coupling probe, the IBRV coupling probe and the BPIV coupling probe of claim 1 with corresponding virus magnetic spheres respectively;
2) Extracting genome nucleic acid of a sample to be detected, and performing PCR amplification detection by using the BVDV primer pair, the IBRV primer pair and the BPIV primer pair in claim 1 by taking the extracted genome nucleic acid as a template;
3) The PCR amplified products are hybridized with the magnetic balls coupled with the virus probes, and hybridization and on-machine analysis of liquid phase chips are carried out, so that detection of bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus is realized;
the PCR reaction system in the step 2) comprises the following steps: one-Step PCR reaction solution 2 XOne Step RT-PCR Buffer III 12.5. Mu.L, taKaRa Ex Taq HS 0.5. Mu.L, primeScript RT Enzyme Mix II 0.5.5. Mu.L, BVDV upstream primer/IBRV upstream primer/BPIV upstream primer each 0.25. Mu.L, BVDV downstream primer/IBRV downstream primer/BPIV downstream primer each 0.5. Mu.L, RNA-free H 2 O7.25. Mu.L, nucleic acid template 2. Mu.L, all primer concentrations of 20 ng/. Mu.L;
the PCR reaction conditions in the step 2) are as follows: firstly, the temperature is 52 ℃ for 15min and 95 ℃ for 2min; then 94 ℃ for 30s,56 ℃ for 30s,72 ℃ for 30s,35 cycles; finally, the final extension is carried out at 72 ℃ for 7min.
6. The method of claim 5, further comprising:
4) And (3) result judgment:
the result judgment criteria were:
A. when the ratio of positive MFI to negative MFI is more than or equal to 3 and positive MFI is more than or equal to 300, the test is established, QC is equal to the ratio of an MFI sample to negative control N-control, namely QC=MFI sample/N-control,
B. when the A is judged to be established in the test, judging that the MFI sample of each virus magnetic sphere is more than or equal to 300, QC is more than or equal to 3, and judging that the virus magnetic sphere is a corresponding virus positive sample; otherwise, judging as negative; namely:
if the MFI value of the magnetic sphere corresponding to the bovine viral diarrhea virus is lower than 300, the sample is free of the bovine viral diarrhea virus, and if the MFI value of the magnetic sphere corresponding to the bovine viral diarrhea virus is more than or equal to 300 and QC is more than or equal to 3, the sample is indicated to contain the bovine viral diarrhea virus; if the MFI value of the magnetic sphere corresponding to the infectious bovine rhinotracheitis virus is lower than 300, the sample is free of the infectious bovine rhinotracheitis virus, and if the MFI value of the magnetic sphere corresponding to the infectious bovine rhinotracheitis virus is more than or equal to 300 and QC is more than or equal to 3, the sample is indicated to contain the infectious bovine rhinotracheitis virus; if the MFI value of the magnetic sphere corresponding to the bovine parainfluenza virus is lower than 300, the sample is free of bovine parainfluenza virus, and if the MFI value of the magnetic sphere corresponding to the bovine parainfluenza virus is more than or equal to 300 and QC is more than or equal to 3, the sample contains bovine parainfluenza virus.
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| CN105671207A (en) * | 2016-03-28 | 2016-06-15 | 上海市农业科学院 | Liquid chip detection method for four types of pathogens of porcine virus diarrhea |
| CN106167837A (en) * | 2016-08-31 | 2016-11-30 | 金宇保灵生物药品有限公司 | The real-time fluorescence quantitative PCR detection kit of bovine viral diarrhoea bovine diarrhoea virus and primer special thereof and probe |
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| CN105671207A (en) * | 2016-03-28 | 2016-06-15 | 上海市农业科学院 | Liquid chip detection method for four types of pathogens of porcine virus diarrhea |
| CN106167837A (en) * | 2016-08-31 | 2016-11-30 | 金宇保灵生物药品有限公司 | The real-time fluorescence quantitative PCR detection kit of bovine viral diarrhoea bovine diarrhoea virus and primer special thereof and probe |
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