CN105274253B - Kit for simultaneously detecting 10 insect-borne pathogens and application thereof - Google Patents
Kit for simultaneously detecting 10 insect-borne pathogens and application thereof Download PDFInfo
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Abstract
The invention provides a kit for simultaneously detecting 10 insect-borne pathogens and application thereof, belonging to the technical field of liquid-phase chip detection. The kit comprises specific primers aiming at 10 common entomopathogenic agents and a probe coupled with a fluorescent labeling microsphere, wherein the nucleotide sequences of the specific primer pairs are respectively shown as SEQ ID NO.1-20, and the nucleotide sequence of the probe is shown as SEQ ID NO. 21-30. The kit provided by the invention optimizes hybridization reaction conditions by setting a multiple asymmetric PCR system, so that accurate detection of 10 entomopathogenic agents is realized. The kit has the advantages of accurate detection, high sensitivity, strong specificity, simplicity, convenience and rapidness, is suitable for port inspection and quarantine and entomopathogenic epidemiological investigation, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of liquid-phase chip detection, in particular to a method for simultaneously detecting 10 entomopathogenic agents by using a liquid-phase chip method, a detection kit and application thereof.
Background
Arboreal disease refers to a disease that is transmitted from blood-sucking arthropods to humans and humans, and to humans and animals by bite infections. The clinical manifestations of the animal are sudden fever, ache and weakness, large area of rash, death caused by secondary infection, etc. The disease causes thousands of people and livestock to be infected and even die in the past, and brings certain negative effects to the development of economy and human society. The continuous development of international animal trade has also been promoted in the middle of the 20 th century with the global increase in traffic, particularly with the use of modern vehicles that have shortened the distance around the world, these factors providing a wider possibility for the long-range spread of arboreal diseases and the tendency to expand the popularity worldwide. At present, with increasingly frequent international communications, the risk of mosquitoes, ticks, midges and other vectors being introduced into the border port of China is very high, and outbreaks of insect-borne animal diseases are also likely to be introduced into China along with biological insect vectors.
More than 100 arboviruses are known to cause epidemic diseases and even death in animals and humans. The most representative includes: mosquito-borne diseases: vesicular Stomatitis (VS), West Nile Fever (West Nile, WN), Rift Valley Fever (RVF); tick-borne diseases: african Swine Fever (ASF), Lyme disease (the etiology is Borrelia burgdorferi, abbreviated as BB), Q Fever (Q Fever, the etiology is coxiella burnetii, also called rickettsia); midge medium disease: bluetongue (BT) and Epizootic Hemorrhagic Disease (EHD), Schmallenberg (Schmallenberg, SB). The epidemic outbreaks of the animal epidemic diseases in peripheral countries in recent years form a threat to China, and particularly, the epidemic situation of Ebola hemorrhagic fever (EBola) in West Africa begins to burst in 2 months in 2014, which causes a great amount of fever and bleeding of people and even death. Therefore, the method for detecting the entomogenous animal epidemic disease pathogen is provided, and particularly, the method for simultaneously detecting various entomogenous animal epidemic disease pathogens has important significance. However, the prior art does not realize simultaneous detection of the 10 entomopathogenic pathogens, and in order to guarantee green development of agriculture, sanitation and social security in China, a rapid, specific, efficient and sensitive method for simultaneously detecting the entomopathogenic pathogens needs to be developed.
The liquid phase chip is called microsphere suspension chip, is a novel biochip technology platform based on xMAP (flexible Multi analysis profiling) technology, and is characterized by that on the microsphere with different fluorescent codes the combination reaction of antigen antibody, enzyme, substrate, ligand and receptor and nucleic acid hybridization reaction can be made, and two beams of red and green laser can be used for respectively detecting microsphere code and reporting fluorescence so as to attain the goal of qualitative and quantitative detection, and one reaction hole can be used for implementing up to 100 different biological reactions, so that it is a new generation high-throughput molecular detection technology platform following gene chip and protein chip.
The liquid phase chip technology platform is a new generation molecular diagnosis technology platform which can ensure the information quality and provide relatively high flux, and the technology platform integrates various advanced technologies such as biological detection, latex microsphere fluorescence coding, a micro-liquid transmission system, laser real-time recording, advanced computer software, a data processing mode and the like. The core technology of the liquid phase chip is to dye the tiny latex microspheres into hundreds of different fluorescent colors (the solid phase chip uses the coordinate position of the probe on the chip to specifically code the gene, and the liquid phase chip uses the color to code). When the method is applied, the latex microspheres for different detection objects are mixed, then a trace of sample to be detected is added, and the sample is specifically combined with the particles in suspension. The combined result can be recorded in the form of data information by a computer after being instantly judged by laser. Since molecular hybridization is carried out in a suspension solution and the detection speed is extremely high, it is called "liquid chip".
The liquid phase chip technology platform has high efficiency: because hundreds of colors of latex microspheres can be in the same reaction system, a small sample (blood or other body fluids, tissues) can be used to simultaneously detect hundreds of physiological or pathological indicators. The liquid phase chip technology platform has high sensitivity: each latex microsphere may be fully coated (by means of a strong covalent bond) with an antigen, antibody, or nucleic acid. Because of the high probe density, the strong signal generated, and the use of fluorescence detection, the sensitivity is much higher than any existing analytical and diagnostic methods, and also higher than other chip methods. The liquid phase chip technology platform is a rapid detection method: because the hybridization is carried out in a suspended liquid phase, the reaction requires a short time, the hybridization can be directly read without washing, the detection efficiency is greatly higher than that of solid phase hybridization, and the time is shortened from a few hours to tens of minutes. In the prior art, a method for detecting entomopathogenic bacteria by adopting a liquid-phase chip method is disclosed in Chinese patent CN102181532A, but 9 kinds of entomopathogenic bacteria targeted by the method are only common entomopathogenic bacteria in China and do not completely relate to overseas epidemic entomopathogenic diseases, however, in inspection and quarantine at the national port, pathogen quarantine is still required to be performed on animals or insects from countries with high incidence of the entomopathogenic diseases, so that the comprehensive prevention and control capability of the China for preventing the remote diffusion of the entomopathogenic diseases of the foreign animals is improved, and domestic animals are protected from being infected by the overseas entomopathogenic bacteria.
Disclosure of Invention
The invention aims to provide a liquid chip method for simultaneously detecting 10 entomopathogenic agents; the invention also aims to provide a kit for simultaneously detecting 10 insect-borne pathogens and application thereof.
To achieve the above object, specific fragments of each insect-borne disease are determined by referring to OIE, national standard, Row standard and related literature, and the corresponding fragments of each insect-borne disease, namely WNV-E gene, VSV-N gene, ASFV-P72 gene, BTV-NS1 gene, Borrelia burgdorferi-OSPA gene, EHDV-NS3 gene, Coxiella burnetii-IS1111a gene (accession number: M80806), SBV-S gene (accession number: HE649914), EBV-NP gene (accession number: KC545392.1), FV-NSs gene (accession number: DQ380154) are downloaded by accessing GenBank. Selecting conserved regions by using DNAMAN, MEGA, BioEdit, DNAStar, Primer Premier5.0 and other software, designing, analyzing and screening 10 pairs of specific primers, 10 specific probes and 10 verification probes (reverse compatibility of probes, RC-probes), labeling the C12 arm with amino modification at the 5 'end of the capture Probe, and modifying biotin at the 5' ends of the downstream primers and the verification probes. Therefore, the invention provides a specific primer pair combination for simultaneously detecting 10 insect-borne pathogens by using a liquid chip, which comprises the following primer pairs:
the nucleotide sequences of the upstream primer and the downstream primer for detecting the African swine fever virus are shown as SEQ ID NO. 1-2;
the nucleotide sequences of the upstream and downstream primers for detecting the bluetongue virus are shown as SEQ ID NO. 3-4;
the nucleotide sequences for detecting the upstream and downstream primers of the Borrelia burgdorferi are shown in SEQ ID NO. 5-6;
the nucleotide sequences of the upstream and downstream primers for detecting the epidemic hemorrhagic fever virus of the deer are shown in SEQ ID NO. 7-8;
the nucleotide sequences of the upstream primer and the downstream primer for detecting the vesicular stomatitis virus are shown as SEQ ID NO. 9-10;
the nucleotide sequences for detecting the upstream and downstream primers of the West Nile fever virus are shown as SEQ ID NO. 11-12;
the nucleotide sequences for detecting the upstream and downstream primers of the coxiella burnetii are shown as SEQ ID NO. 13-14;
the nucleotide sequences of the upstream and downstream primers for detecting rift valley fever virus are shown in SEQ ID NO. 15-16;
the nucleotide sequences for detecting the upstream and downstream primers of the Ebola virus are shown as SEQ ID NO. 17-18;
the nucleotide sequences for detecting the upstream and downstream primers of the Schmallenberg virus are shown as SEQ ID NO. 19-20.
The 5' end of the downstream primer is labeled with biotin.
Further, the present invention provides a specific probe set used in combination with the specific primer set described above, comprising the following probes:
the nucleotide sequence of the probe for detecting the African swine fever virus is shown as SEQ ID NO. 21;
the nucleotide sequence of the probe for detecting the bluetongue virus is shown as SEQ ID NO. 22;
the nucleotide sequence of the probe for detecting the borrelia burgdorferi is shown as SEQ ID NO. 23;
the nucleotide sequence of the probe for detecting the deer epidemic hemorrhagic fever virus is shown as SEQ ID NO. 24;
the nucleotide sequence of a probe for detecting the vesicular stomatitis virus is shown as SEQ ID NO. 25;
the nucleotide sequence of the probe for detecting the West Nile fever virus is shown as SEQ ID NO. 26;
the nucleotide sequence of a probe for detecting coxiella burnetii is shown as SEQ ID NO. 27;
the nucleotide sequence of the probe for detecting rift valley fever virus is shown in SEQ ID NO. 28;
the nucleotide sequence of the probe for detecting the Ebola virus is shown as SEQ ID NO. 29;
the nucleotide sequence of the probe for detecting the Schmallenberg virus is shown as SEQ ID NO. 30.
C in which the probe is amino-modified at its 5' end12Marking of the arm.
The invention provides a liquid-phase chip, which comprises 10 specific detection microspheres prepared by respectively coupling 10 probes in the 10 specific probe combinations with fluorescence labeling microspheres.
The invention provides application of the 10 pairs of specific primer pairs and 10 specific probes in preparation of a kit for simultaneously detecting 10 insect-mediated pathogens.
The invention also provides a kit for simultaneously detecting 10 entomopathogenic agents, which comprises the 10 pairs of specific primer pairs and 10 specific detection microsphere liquid-phase chips prepared by respectively coupling the 10 probes and fluorescent labeled microspheres. The 10 insect-borne diseases are respectively as follows: african swine fever, bluetongue, Lyme disease, epizootic hemorrhagic fever, vesicular stomatitis, West Nile fever, Q fever, rift valley fever, Ebola and Schmallenberg disease.
The working procedure of the kit capable of simultaneously detecting 10 insect-mediated pathogens provided by the invention comprises the following steps:
(1) extracting nucleic acid of a sample to be detected;
(2) carrying out RT-PCR reaction on the RNA or DNA of the sample by using the 10 pairs of specific primer pairs aiming at the 10 entomopathogenic bacteria;
(3) after molecular hybridization is carried out on the PCR product with the biotin label and the liquid phase chip, the fluorescence value is detected, and whether the sample contains African swine fever virus, bluetongue virus, borrelia burgdorferi, deer epidemic hemorrhagic fever virus, vesicular stomatitis virus, West Nile fever virus, Coxiella burnetii, rift valley fever virus, Ebola virus and/or Schmallenberg virus is determined.
In the RT-PCR reaction system in the step (2), a multiple asymmetric PCR system is arranged, and the result shows that the optimal detection effect can be achieved when the concentration ratio of the upstream primer to the downstream primer of each pair of primers in the specific primer pair combination is 1: 2.
In one embodiment of the present invention, the RT-PCR reaction of step (2) is performed in a 50. mu.L reaction system: 23. mu.L of RT-PCRmix buffer, 5.0. mu.L of RNA template, 0.5. mu.L of 10 upstream primers, 1.0. mu.L of 10 downstream primers, and ddH containing no RNase2O is complemented to 50 mu L; the PCR reaction conditions are as follows: 30min at 50 ℃; 15min at 95 ℃; 35 cycles of 95 ℃ for 40s, 54 ℃ for 40s and 72 ℃ for 1 min; 10min at 72 ℃.
And (3) configuring a 50 mu L hybridization system consisting of the PCR product and the liquid chip as follows: 33 mu L of microsphere mixed solution, 15 mu L of TE Buffer with pH of 8.0 and 2 mu L of positive PCR product; the microsphere mixed solution is prepared by adding 1 mu L of 10 fluorescence labeling coupled microspheres in each 330 mu L of 1 XTMAC hybridization buffer solution.
The hybridization conditions of the step (3) are as follows: denaturation at 95 ℃ for 5min, and hybridization at 50 ℃ for 30 min.
The primers and the probes for detecting the 10 entomopathogenic bacteria are designed aiming at the 10 entomopathogenic bacteria, so that the primers and the probes are prevented from forming a secondary structure, and mismatching and non-specific fragments are prevented; in the multiple liquid phase chip, the TM value of the probe directly influences the hybridization temperature, when designing the primer and the probe sequence, the TM values of the upstream primer and the downstream primer and the TM values among the probes are considered to be as close as possible, amino modification is carried out at the 5' end of the probe, and the amino modification reacts with a large amount of carboxyl on the surface of the magnetic microsphere through EDC action to form a probe and microsphere compound with the temperature, so that the hybridization efficiency is improved. In addition, in the design process, the invention also designs a probe which is reverse complementary to the probe, namely a verification probe, aiming at different probes, and carries out 5' biotin labeling for verifying the coupling efficiency of the probe and the microsphere, and the final result shows that the probe and the microsphere realize the most efficient coupling.
The invention optimizes the configuration of a hybridization reaction system, the temperature, the time and the like of the hybridization reaction by arranging a multiple asymmetric PCR system, thereby realizing the accurate detection of 10 entomopathogenic diseases. The kit has the advantages of accurate detection, high sensitivity, strong specificity, simplicity, convenience and rapidness, is suitable for port inspection and quarantine and entomopathogenic epidemiological investigation, and has good application value.
Drawings
FIG. 1 is a diagram showing the results of optimizing the ratio of upstream and downstream primers in an asymmetric PCR reaction. In order to ensure that the maximum amount of biotin-modified single-stranded nucleic acid is obtained, PCR amplification reaction is simultaneously carried out according to the concentrations of upstream and downstream primers with different proportions in 10-fold asymmetric PCR reaction, and the obtained fluorescence value is very high when the upstream and downstream primer proportions are 1:2, 1:3 and 1: 4.
FIG. 2 is a diagram showing the optimization results of the amount of PCR products used in the hybridization reaction. In the hybridization reaction, 6 PCR products diluted in a gradient of 0.5. mu.l, 1. mu.l, 2. mu.l, 4. mu.l, 6. mu.l, 8. mu.l, etc. were added, and as a result, it was found that the fluorescence value reached the maximum when 2. mu.l of the PCR product was added, and then the fluorescence value decreased with the increase of the product, and therefore, the amount of the PCR product was finally selected to be 2. mu.l, and each set of the bar graphs in the figure was WNV, BB, ASFV, VSV, BTV, EHDV, QF, EBV, RVFV, SBV, respectively, from the left and the.
FIG. 3 is a graph showing the results of optimization of hybridization temperature in hybridization reaction. In order to determine the optimal hybridization temperature, 6 temperatures of 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃ and the like are selected for hybridization, and the result shows that the highest hybridization efficiency is obtained when the hybridization temperature is 50 ℃, and the histogram of each temperature group in the graph is WNV, BB, ASFV, VSV, BTV, EHDV, QF, EBV, RVFV and SBV from the left and the right respectively.
FIG. 4 is a graph showing the results of optimization of hybridization time in hybridization reaction. In order to obtain the best hybridization efficiency, hybridization was carried out for 15, 20, 25, 30, 35 and 40min according to other optimized conditions, and the results showed that the maximum fluorescence detection signal was obtained at 30min of hybridization.
FIG. 5 is a graph showing the results of the specificity test. The result of the kit assembled by the invention shows that the kit can only specifically detect 10 corresponding pathogens without cross reaction with other common diseases for 10 kinds of entomovirus nucleic acids, and common other animal pathogens, namely peste des petits ruminants virus (PPRV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Foot and Mouth Disease Virus (FMDV) and Brucella.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; all reagents used in the examples are commercially available unless otherwise specified.
EXAMPLE 1 design of primers and probes
Specific fragments for each insect-borne disease were determined by consulting OIE, national standard, Row standard and related literature, and the GenBanK was registered, and the corresponding fragments for each insect-borne disease, i.e., WNV-E gene, VSV-N gene, ASFV-P72 gene, BTV-NS1 gene, Borrelia burgdorferi-OSPA gene, EHDV-NS3 gene, Coxiella burnetii-IS1111a gene (accession number: M80806), SBV-S gene (accession number: HE649914), EBV-NP gene (accession number: KC545392.1), RVFVV-NSs gene (accession number: DQ380154), were downloaded. The gene sequences of the 10 entomopathogenic agents are analyzed and compared by using software such as DNAMAN, MEGA, BioEdit, DNAStar, Primer Premier5.0 and the like, conserved regions are selected, 10 pairs of specific primers, 10 specific probes and 10 verification probes (RC-probes) are repeatedly researched and tested for many times, 10 pairs of specific primers, 10 pairs of specific probes and 10 pairs of verification probes are designed, analyzed and screened, the 5 'end of the capture Probe is marked by an amino-modified C12 arm, and the 5' ends of the downstream primers and the verification probes are modified by biotin. The designed primers and probes were aligned and analyzed by NCBI PrimerBLAST and showed good specificity with the related sequence BLAST in NCBI, and all probes were aligned and analyzed by Clusta W and showed homology of less than 40%. And synthesized by Shanghai Biotech, Inc., with the sequences as shown in tables 1 and 2.
TABLE 1 primer sequences
TABLE 2 Probe sequences
The design of the primer and the probe is a key factor of a liquid phase chip, so when the primer and the probe are designed, firstly, the primer with higher specificity is selected from nucleotide fragments with high conservation, but when the primer is designed, not only are highly conserved regions among different types of each entomopathogenic agent considered, but also specific fragments of certain entomopathogenic agents can be simultaneously represented in the conserved regions, and the specificity among the highly conserved regions is also ensured, so that different entomopathogenic fragments can be easily distinguished. For example, EHDV has 24 genotypes, and the 24 genotypes need to be analyzed simultaneously, the upstream and downstream primers are screened and the probe fragment is analyzed to ensure that the EHDV is distinguished from other insect-borne pathogens and the homology does not exceed 50%. In addition, if the variation of different VSVs is large, a conservative probe needs to be designed first, and a specific primer needs to be designed on the basis of the probe.
(2) Secondary structures are prevented from forming among the primers, such as a self hairpin structure, an upstream primer and a downstream primer, or a dimer is formed between the primers and the probes, and the primers are prevented from being mismatched with the target gene, so that non-specific fragments can be generated if one primer is combined with multiple places of the target gene. In the liquid phase hybridization, the probe is prevented from being mismatched with a target gene segment, particularly with a biotin-labeled downstream primer, so that the normal hybridization is competitively inhibited, and false positive or false negative is formed;
(3) the 3 'end of the primer and the 5' end of the probe are required to be highly conserved, in the annealing process, the 3 end of the primer is the place for starting extension, so that the most efficient replication can be carried out, and in the probe coupling link, the 5 end amino of the probe and a large amount of carboxyl on the surface of the magnetic microsphere form a stable probe-microsphere complex through the action of EDC.
(4) The Tm value is an important determinant factor for designing the primer and the probe, provides reference data for the optimal annealing temperature of the primer, the Tm values of the upstream primer and the downstream primer are close to each other as possible, simultaneously, the Tm values of all the upstream primer and the downstream primer of 10 entomogenous pathogens are considered not to exceed 10 ℃ at most, preparation is made for multiplex PCR, in addition, in order to improve the hybridization efficiency in a multiple liquid phase chip, the Tm values of the probe have great influence, the Tm values of various probes are kept close to each other as much as possible, and the difference value between the Tm values of various probes exceeds 4 ℃.
(5) Because of a large number of pathogens detected simultaneously, multiple pairs of primers and multiple probes need to be designed for each insect-borne disease, cross reactions among 10 pairs of primers, between a primer pair and a template, between probes and different PCR products are avoided, and finally, the optimal primer pairs and probes in tables 1 and 2 are screened out.
Example 2 optimization of multiplex PCR detection System for simultaneously detecting 10 insect-mediated pathogens
1. Extraction of entomopathogenic RNA extraction was performed using the QIAamp Viral RNAMini kit, see kit instructions for procedures.
2. Establishment of multiple asymmetric PCR System
The conditions such as dNTP concentration, enzyme amount, annealing temperature and the like are optimized, and particularly, the repeated optimization of the concentration ratio of the upstream primer and the downstream primer is emphasized. Because the downstream primer is labeled by Biotin, the single-stranded DNA with a Biotin label is improved, thereby improving the hybridization efficiency.
In the asymmetric PCR reaction, PCR reaction is carried out according to the concentration ratio of upstream and downstream primers (1:1, 1:2, 1:3, 1:4, 1: 5), then hybridization detection is carried out, and the optimal upstream and downstream primer concentration ratio suitable for single-fold and multiple-fold asymmetric PCR reaction is further determined.
In order to ensure that the maximum amount of biotin-modified single-stranded nucleic acid is obtained, PCR amplification reaction is simultaneously carried out according to the concentrations of upstream and downstream primers with different proportions in 10-fold asymmetric PCR reaction, detection of 10-fold liquid phase chip method is carried out under the condition that other systems and conditions are not changed, and then the optimal concentrations of the upstream and downstream primers are determined. The results show that the number of readings of 10 different coded magnetic fluorescent microspheres is not less than 100, and the blank control and the negative reference both have values less than 300, the results indicate that the counted amount of microspheres is effective and the resulting fluorescence value can be analyzed, the test results can be determined, the results are shown in FIG. 1, and it can be seen from the figure that median fluorescence values of RVFV, SBV, EBV, WNV and BTV are the highest at the ratio of 1:2, 1:3 and 1:4, and basically reach more than 4000, the fluorescence values are high and even approach to 10000, the median fluorescence intensity values of BB and ASFV in the system are relatively stable, EHDV, VSV and QF have the best median fluorescence intensity values when the upstream and downstream primers are 1:2, considering that the cost is reduced in the experiment, the best effect is achieved by the smallest amount as possible, and the limitation of the concentration conditions of the EHDV, VSV and QF upstream and downstream primers, it was concluded that setting the proportional upstream and downstream primer concentrations to 1:2 was sufficient to achieve good detection results.
Thus, in this example, the multiplex asymmetric PCR reaction system was determined as follows: mix buffer 23. mu.L, RNA template 5.0. mu.L, forward primer 10. mu. mol/L0.5. mu.L each, reverse primer 10. mu. mol/L1.0. mu.L each, ddH without RNase2O complement to 50 μ LPCR amplification reaction program: 30min at 50 ℃; 15min at 95 ℃; 95 deg.C40s, 54 ℃ for 40s, 72 ℃ for 1min (35 cycles); extending for 10min at 72 ℃, and storing at 4 ℃.
EXAMPLE 3 preparation of liquid phase chip
1. Coupling of probes to microspheres
Coupling a WNV probe and a #27 microsphere, a BB probe and a #28 microsphere, an ASFV probe and a #26 microsphere, a VSV probe and a #64 microsphere, a BTV probe and a #35 microsphere, a Q Fever probe and a #43 microsphere, an RVFV probe and a #47 microsphere, an EBV probe and a #21 microsphere, an EHDV probe and a #52 microsphere and an SBV probe and a #68 microsphere according to the coupling steps of the probe microspheres, and specifically comprises the following steps:
(1) 2 fresh EDC powders were initially taken off and left to stand at room temperature (about 1 h).
(2) 10 microspheres (concentration 1.25X 10) of different colors were taken out7pieces/mL) was allowed to stand at room temperature and vortexed for 30 seconds to suspend the microspheres thoroughly and uniformly.
(3) 200. mu.L of each of the mixed microspheres was put into a 1.5ml brown EP tube, centrifuged at 14000g for 3 to 5min, and then put into Invitrogen Bead Separation (magnetic Bead separator), briefly held, and the supernatant was aspirated.
(4) Add 50. mu.L of 0.1M MES (pH 4.5) to resuspend the microspheres, shake for 20-30s, and then sonicate the microspheres for 20s with a sonicator.
(5) Each of 10 different probes (non-verification probes) 3. mu.L (stock solution with a concentration of 0.1 nmol/. mu.L) described in Table 2 of example 1 were added to the microspheres, and the mixture was shaken and mixed to sonicate the microspheres for 20 seconds.
(6) 2.5. mu.L of freshly prepared EDC solution (10mg/mL) was added to each brown EP tube reaction, vortexed quickly and mixed well, and left at room temperature in the dark for 30min, noting that the EDC solution was prepared with molecular biological water and the shelf life was 30 min.
(7) Add 2.5. mu.L of freshly prepared EDC solution (10mg/mL) to each reaction, vortex rapidly and mix well, and leave in the dark at room temperature for 30 min.
(8) To each reaction 1ml of 0.02% Tween-20 solution was added, vortexed briefly, centrifuged at full speed for 2min, placed in a magnetic rack, and the supernatant aspirated.
(9) 1ml of 0.1% SDS solution was added to the reaction to wash the microspheres, the mixture was shaken for 40 seconds, centrifuged at full speed for 2min and then placed in a magnetic bead separator, and the supernatant was aspirated.
(10) Resuspending the microspheres in 100. mu.LTE solution (pH 8.0), vortexing and sonicating for 30s each, and storing at 4 ℃ for future use
(11) Diluting 1 μ L of microsphere by 100 times, shaking, mixing, and counting 10 μ L of microsphere diluent on a hemocytometer, wherein the number of each microsphere is required to be not less than 5000.
2. Verification of coupling efficiency of probe and microsphere
(1) The stored concentration of 100. mu.M (i.e., 100 pmol/. mu.L) of the validation Probe (rc-Probe-Biotion) was first removed, and the stored concentration was diluted in a 10-fold gradient to 10 pmol/. mu.L, 1 pmol/. mu.L, 100 fmol/. mu.L, 10 fmol/. mu.L, and 1 fmol/. mu.L of the validation Probe at different concentrations, as shown in Table 2 in example 1, and placed on ice for use.
(2) The corresponding microspheres coupled with the probes in step 1 of this example were taken out, and subjected to shaking, mixing and sonication for 30 seconds each.
(3) Each set of microspheres was diluted with 1 XTAC hybridization solution to 1. mu.L of a mixture of not less than 100 beads.
(4) In a standard PCR eight-row tube, adding 1. mu.l of biotin-labeled verification probes with different concentrations, a microsphere mixed solution and TE Buffer according to the table 3, fully and uniformly mixing, and making 3 multiple holes.
TABLE 3 addition amounts of probes, microsphere mixture and TE Buffer
(5) Placing the PCR octal row tube in a PCR instrument, denaturing at 95 ℃ for 5min, hybridizing at 54 ℃ for 30min, and keeping at 4 ℃.
(6) And transferring the reaction liquid in the PCR eight-row tube to a 96-hole ELISA plate, paying attention to avoid generating bubbles, placing the plate on a magnetic frame to adsorb magnetic microspheres, and discarding the liquid.
(7) Simultaneously, the 1 XTMAC hybridization solution was subjected to 1000: the 3-fold dilution was dispensed and approximately 100. mu.L of reporter buffer resuspended microspheres per well.
(8) The ELISA plates were sealed with aluminum foil and incubated in a metal bath at 58 ℃ for 10min in the dark.
(9) The ELISA plate is put into a Bio-Plex liquid phase chip detection system which is preheated, corrected and checked well in advance for analysis, the size of a detection sample is adjusted to be 100ul, the range of the detection sample is adjusted to be 100 microspheres, and finally the coupling efficiency of the microspheres and the probes is judged according to a median fluorescence intensity value (MFI).
In the method, a verification probe which is reversely complementary with the probe and labeled by biotin is used as a positive probe, and the verification probe is hybridized with a capture probe coupled with the microsphere after being diluted by 0 fmol-1000 fmol concentration to detect the coupling efficiency. The result shows that the verification probes aiming at 10 genes and the respective capture probes have higher hybridization signals, and the median value of the fluorescence intensity gradually decreases from high to low along with the concentration of the verification probes to present a certain gradient, which indicates that the coupling of the probes and the microspheres is successful.
Example 4 optimization of hybridization System and reaction conditions
1. Hybridization assay
Performing liquid phase hybridization detection on the PCR product of the arbovirus obtained in the example 2 and the microspheres coupled with the virus probes prepared in the example 3, and designing a blank control and a negative control at the same time, wherein the steps are as follows:
(1) selecting 10 different microsphere probes for coupling combination, and respectively carrying out vortex and ultrasonic treatment for 30s to ensure that the microspheres are uniformly suspended.
(2) Preparing a mixed microsphere working solution: add 10 coupled microspheres 1. mu.l each per 330ul of 1 XTMAC hybridization buffer, and shake and sonicate for 30s each to mix the working solution.
(3) To each reaction tube in the PCR octa-row, 33. mu.L of the mixed microsphere working solution and 16. mu.L of TEBuffer (pH 8.0) were added, respectively. Add 1ul ddH to blank control tube2O; adding 1 mu L of PCR negative amplification product into a negative control tube; adding 1 mu L (the concentration is 0.01 mu mol/L) of verification probes corresponding to the microsphere coupling probe combination into the positive control tube; add 1. mu.L of positive PCR products into the sample detection tube, make 3 multiple wells each, then blow gently with pipette, mix well.
(4) The tubes were placed in a PCR instrument and the PCR product was denatured at 95 ℃ for 5min, hybridized at 54 ℃ for 30min and maintained at 4 ℃.
(5) And transferring the hybridization reaction liquid in the PCR eight-row calandria to a 96-hole ELISA plate, taking care to avoid generating bubbles, placing the plate on a magnetic frame to adsorb the magnetic microspheres for about 1min, and discarding the liquid.
(6) Simultaneously, the 1 XTMAC hybridization solution was subjected to 1000: the 3-fold dilution was dispensed at a final concentration of 3pg/mL, and approximately 100. mu.L of reporter buffer resuspended microspheres were added per well.
(7) The ELISA plates were sealed with aluminum foil to prevent evaporation of the liquid and incubated in a metal bath at 58 ℃ for 10min in the dark.
(8) The ELISA plate is placed into a Bio-Plex liquid phase chip detection system which is preheated, corrected and checked well in advance to analyze 100 mul of samples, the size of the detected samples is adjusted to be 100 mul and the range is 100 microspheres, and finally the result is judged according to the median fluorescence intensity value (MFI) read by each hole and the judgment standard.
2. Use and result judgment standard of liquid-phase chip detector
The liquid phase chip detection operation was performed according to the use course of the suspension chip detection system recommended by Bio-Rad, USA. According to the judgment standard recommended by Bio-Rad company, when the number of the magnetic fluorescent coding microspheres is larger than or equal to 100, a group of data point communities are generated in the white areas of the corresponding microsphere labels, and the background blank fluorescence intensity value is smaller than 500, which indicates that the experiment is established, and the result judgment can be carried out. The liquid phase chip qualitative ratio result (LQRR) is equal to the ratio of the corrected Median Fluorescence Intensity (MFIS) of the sample to the mean MFIB of the blank MFI, i.e., LQRR is MFIS/MFIB. If LQRR is more than or equal to 3, determining as a positive sample; if LQRR is more than or equal to 2 and less than 3, the detection is judged to be suspicious and needs to be detected again; if LQRR <2, then the result is judged to be negative. According to the judgment standard, when the fluorescence value of the blank control is less than 500, the average fluorescence value of the detection sample is more than 3 times of that of the blank control, and the detection sample is judged to be positive; less than 2 times negative; if the ratio is more than 2 times and less than 3 times, the result is judged to be suspicious and needs to be rechecked. 3. Optimization of hybridization conditions for suspension chip systems
As the 5.6 μm magnetic fluorescent coded microspheres used in this example were purchased directly and the coupling of the capture probes and microspheres at the previous stage was performed by referring to the method disclosed by Bio-Rad, the important step in the experimental design is the hybridization of the PCR product with the coupled probes of the microspheres, i.e., the optimization of various hybridization conditions is focused. The optimal conditions for the hybridization of the suspension chip can improve the sensitivity of the hybridization, ensure the specificity of the hybridization and prevent the occurrence of false positive and false negative. This example was optimized for each of the conditions listed below that may affect the hybridization results.
3.1 optimization of the amount of PCR product in the hybridization reaction
In the hybridization annealing link, a large number of capture oligonucleotide probe molecules are covalently connected to the surface of the magnetic microsphere, and excessive or insufficient PCR product amount can generate competitive interference on the surface of the microsphere and insufficient capture force, so that false negative and false positive are easily caused, the PCR product amount has obvious influence on the hybridization efficiency, and the optimal PCR product volume amount achieves the highest hybridization efficiency by using the fewest PCR products. Under the condition that the concentration ratio of the primers is determined to be 1:2, other conditions and the system are not changed, in a liquid phase hybridization reaction system, probes of 10 different entomoviruses are simultaneously used as a research object, the amount of a mixed PCR product amplified by asymmetric PCR is optimized, 6 gradient diluted PCR product amounts of 0.5 mu L, 1 mu L, 2 mu L, 4 mu L, 6 mu L, 8 mu L and the like are selected, and the influence of PCR products with different volumes in the system on the detection effect of a liquid phase chip is inspected. The best detection effect is that the median fluorescence intensity values corresponding to 10 arboviruses can be simultaneously detected by using the least PCR products, and the negative and positive are easily distinguished. The result shows that the number of the read magnetic fluorescent microspheres with 10 different codes is not less than 100, and the values of the blank control and the negative reference are less than 300, the result shows that the counted microsphere amount is effective, the generated fluorescence value is credible, and the test result can be judged. The results are shown in FIG. 2. As can be seen from the figure, the fluorescence value reaches the maximum when the BTV, EHDV, QF, EBV and ASFV PCR products are 2 muL, the fluorescence value is decreased with the increase of the products, the fluorescence values of WNV, BB, VSV, RVFV and SBV are increased with the volume amount of the PCR products in a certain range, the fluorescence value is basically unchanged when the volume amount of the 2 muL products is increased, when the detection of 10 entomopathogenic agents is considered integrally, the excessive and small amount of the PCR volume can generate certain influence, and the optimal PCR product is selected to be 2 muL for uniformity.
3.2 optimization of hybridization temperature
The hybridization temperature has obvious influence on the hybridization efficiency, the steric hindrance of microspheres in a suspension phase is small, a probe can capture own target genes in a short time, the interference of double chains is eliminated, and a stable hybridization acting force is formed. In the present study, 10 different probes were used as the study targets, and 6 temperature gradients, such as 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃ and the like, were selected as the study targets according to the designed Tm values of the 10 insect-mediated probes, and the hybridization efficiency of different temperatures on the liquid-phase chip was examined under the condition that other conditions were not changed.
The results show that the number of readings of 10 different coded magnetic fluorescent microspheres is not less than 100, and the values of the blank control and the negative reference are less than 300, and the results show that the counted microsphere amount is effective, and the generated fluorescence value can be judged. As shown in FIG. 3, the fluorescence values of BB, EBV and RVFV were kept constant at about 2500, 5000 and 3000 in the range of 50 ℃ to 60 ℃ and the fluorescence values of the remaining 7 arboviruses were gradually decreased with increasing temperature, so that the optimal hybridization temperature was 50 ℃ while 10-fold hybridization was performed.
3.3 optimization of hybridization time
In order to achieve the optimal hybridization efficiency in a short time, under the condition that other conditions and systems are not changed, the hybridization time is selected from 15min, 20min, 25min, 30min, 35min and 40min, so that the hybridization efficiency of the liquid phase chip under different hybridization time is examined, and the optimal hybridization time is determined. Optimizing the hybridization time, in the process of liquid phase hybridization, amplifying PCR by 1:2 upstream and downstream primer concentrations, adding 2ul of mixed PCR products into a hybridization system, setting the hybridization annealing temperature to be 50 ℃, repeating 6 hybridization systems, and respectively hybridizing for 15min, 20min, 25min, 30min, 35min and 40min to determine the optimal hybridization time. The results show that the number of readings of 10 different coded magnetic fluorescent microspheres is not less than 100, and the values of the blank control and the negative reference are less than 300, and the results show that the counted microsphere amount is effective, and the generated fluorescence value can be judged. As shown in FIG. 4, the MFI values of BB, EHDV, QF and SBV are substantially balanced and at the maximum value in 30min and 35min, and the maximum fluorescence detection signals are obtained when the remaining 7 arboviruses are hybridized at 30min, and if the maximum fluorescence intensity value is obtained, the hybridization can be carried out at 50 ℃ for 30 min.
EXAMPLE 5 establishment of liquid chip assay for the method of simultaneously detecting 10 insect-mediated pathogens
1. According to comprehensive analysis and by combining the optimized optimal conditions, determining a detection system and conditions of the 10-recombinant arbovirus liquid phase chip:
nucleic acids of a sample to be tested were extracted, and PCR was performed using 10 pairs of specific primers in Table 1 of example 1.
23. mu.L of one-step RT-PCR mix buffer, 5.0. mu.L of RNA template, 0.5. mu.L of each 10. mu. mol/L upstream primer, 1.0. mu.L of each 10. mu. mol/L downstream primer, ddH without RNase2O complement to 50 μ L PCR amplification reaction program: 30min at 50 ℃; 15min at 95 ℃; 95 ℃ 40s, 54 ℃ 40s, 72 ℃ 1min (35 cycles); extending for 10min at 72 ℃, and storing at 4 ℃.
Liquid phase chip hybridization reaction system: mu.l of microsphere mixed solution (1. mu.l of each of the 1 XTMAC hybridization buffer solution and 10 probes coupled with the fluorescence labeling microspheres is added to 330. mu.l of the 1 XTMAC hybridization buffer solution, 1. mu.l of each of the 10 fluorescence labeling coupled microspheres is prepared according to the method in example 3), 15. mu.l of TEBuffer (pH 8.0) and 2. mu.l of the positive PCR product are denatured for 5min at 95 ℃ in a PCR instrument, the hybridization temperature is 50 ℃, the time is 30min, after the hybridization is finished, an ELISA plate is transferred, 100. mu.l of report buffer solution (3g/mL) is added to each well, the incubation is carried out for 10min at 58. Because different probes are coupled with different microspheres, each microsphere has different colors, a detection machine automatically distinguishes the microspheres, for example, the African swine fever coupled microspheres are 26, the instrument firstly finds the 26 microspheres, and then detects fluorescence values carried by all the 26 microspheres due to hybridization reaction. Therefore, if 10 microspheres are put together, the machine automatically distinguishes different diseases according to the number of the microspheres, and then judges whether the microspheres are positive or negative according to the fluorescence value after reaction.
And (4) judging the standard: according to the judgment standard recommended by Bio-Rad company, when the number of the magnetic fluorescent coding microspheres is more than or equal to 100 and the background blank fluorescence intensity value is less than 500, the experiment is established, and the result judgment can be carried out. The qualitative ratio result (LQRR) of the liquid phase chip is equal to the corrected Median Fluorescence Intensity (MFI) of the sampleS) Mean value of MFI with blank control (MFI)B) I.e. LQRR-MFIS/MFIB. If LQRR is more than or equal to 3, determining as a positive sample; if 2 is less than or equal to LQRR<3, judging the test result to be suspicious and needing to be detected again; if LQRR<And 2, judging the test result to be negative.
2. Authentication
On the basis of the establishment of the 10-fold liquid phase chip detection system, RNA of BTV, EHDV, QF, EBV, ASFV, WNV, BB, VSV, RVFV and SBV is subjected to multiplex amplification and liquid phase chip detection according to the method, the result shows that the number of read 10 different coded magnetic fluorescent microspheres is more than 100, the values of blank control and negative reference are both less than 300, the counted microsphere amount is effective, the result can be judged, the ratio of the fluorescence intensity median of each arbovirus to the MFI of the blank control is both more than 3, and the result shows that the 10-fold liquid phase detection of the arbovirus nucleic acid is positive.
Example 6 characterization of the method of the invention
1. Specificity test
The specificity of the chip method of the entomogenous virus suspension is based on the specificity of respective entomogenous virus primer and the specificity of the probe coupled with the microsphere in PCR amplification, 10 kinds of entomogenous virus nucleic acid are respectively used as templates, common animal epidemic diseases PPRV, PRRSV, FMDV and Brucella (Brucella) are simultaneously utilized to check whether cross infection exists, then 10 designed pairs of primers are used for PCR asymmetric amplification, and then 10 probes are simultaneously used for detecting the specific fluorescence signal intensity by using a liquid phase chip. The results show that the number of all microspheres is not less than 100, and the values of the negative reference and the blank control are both less than 300, which indicates that the detected fluorescence signals are effective, as shown in FIG. 5, the specific primers can be used for amplifying the corresponding positive target fragments, the probes coupled with the microspheres can capture the respective positive PCR products, the probes do not non-specifically hybridize with other probes, the detection results of the probes in liquid phase are positive for 10 kinds of arboviruses and accord with the detection of PCR electrophoresis, and the detection values of the PCR amplification products of the non-target fragments are negative and have little cross reaction. Therefore, the established detection method of the 10-weight insect-medium liquid-phase chip has high specificity, and the primer and the probe have good specificity, so that the method is established.
2. Sensitivity test
Deionized water unifies the concentration of the 10 arbovirus nucleic acids to the same order of magnitude of 1010copy number for future use and 10% sterilized ultrapure water1、102、103、104、105、106、107、108、109And (5) diluting by times. Asymmetric PCR amplification is carried out by taking the different concentrations as templates, and the amplification results are respectively detected by liquid chip hybridization and analyzed by gel electrophoresis, so as to determine the sensitivity of the detection method. 5 times of gel electrophoresis analysis is respectively carried out, and 10 insect vectors can be detected4-105Copy number, with individual viruses considered at 103-102The number of copies is suspected, but the definition is not enough, and the judgment cannot be carried out. The detection result of the liquid phase chip is shown in the figure, the median fluorescence value is gradually reduced along with the gradual reduction of the copy number of the 10 kinds of the entomoviruses, and the fluorescence value is diluted to 102When the copy number value is larger than 3, the LQRRs are judged to be positive, and 10 can be detected by BB, EBV and SBV1The copy number and the result show that the sensitivity of the liquid phase chip detection method is obviously higher than that of the PCR method, and the minimum is 10 of the common PCR2And the sensitivity is high.
3. Repeatability test
3 liquid phase chip specificity repeated tests carried out on 10 arbovirus RT-PCR specific products show that 3 detection qualitative results are 100% consistent, and the variation coefficient of each target gene detection is within 10% (CV is standard deviation/arithmetic mean value multiplied by 100%), which shows that the 10 liquid phase chip detection method has good repeatability.
TABLE 4
Example 7 detection of clinical samples by the method of the invention
300 parts of nucleic acid from mosquitoes, ticks and midges provided by each entry and exit quarantine bureau are extracted and blindly detected by using the insect-mediated multiple liquid-phase chip system established in example 5, wherein 6 parts of Borrelia Burgdorferi (BB) in ticks are detected, and the detection is carried out again by using a borrelia burgdorferi nucleic acid detection kit (PCR-fluorescent probe method) provided by Wei burgxin biotechnology limited, Guangzhou, and the detection result is consistent with 100 percent of the detection result of the kit. 13 parts of bluetongue virus (BTV) carried in the mosquitoes are detected, and the further detection is carried out by using a bluetongue virus fluorescence RT-PCR detection kit provided by Beijing Conson biotechnology development Limited company, and the result accords with the method of the invention. 7 positive samples of coxiella burnetii nucleic acid were also detected in the unknown samples. The detection result of the field sample shows that the 10-fold liquid phase chip detection method has good clinical detection result and is suitable for clinical popularization and application of various entry and exit inspection and quarantine bureaus.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (2)
1. The application of a specific primer probe combination in preparing a kit for simultaneously detecting 10 insect-borne pathogens is characterized in that the specific primer probe combination consists of 10 pairs of primers and 10 probes, and the primers and the probes respectively comprise:
the nucleotide sequences of the upstream primer and the downstream primer for detecting the African swine fever virus are shown as SEQ ID NO. 1-2;
the nucleotide sequences of the upstream and downstream primers for detecting the bluetongue virus are shown as SEQ ID NO. 3-4;
the nucleotide sequences for detecting the upstream and downstream primers of the Borrelia burgdorferi are shown in SEQ ID NO. 5-6;
the nucleotide sequences of the upstream and downstream primers for detecting the epidemic hemorrhagic fever virus of the deer are shown in SEQ ID NO. 7-8;
the nucleotide sequences of the upstream primer and the downstream primer for detecting the vesicular stomatitis virus are shown as SEQ ID NO. 9-10;
the nucleotide sequences for detecting the upstream and downstream primers of the West Nile fever virus are shown as SEQ ID NO. 11-12;
the nucleotide sequences for detecting the upstream and downstream primers of the coxiella burnetii are shown as SEQ ID NO. 13-14;
the nucleotide sequences of the upstream and downstream primers for detecting rift valley fever virus are shown in SEQ ID NO. 15-16;
the nucleotide sequences for detecting the upstream and downstream primers of the Ebola virus are shown as SEQ ID NO. 17-18;
the nucleotide sequences for detecting the upstream and downstream primers of the Schmallenberg virus are shown as SEQ ID NO. 19-20;
the nucleotide sequence of the probe for detecting the African swine fever virus is shown as SEQ ID NO. 21;
the nucleotide sequence of the probe for detecting the bluetongue virus is shown as SEQID No. 22;
the nucleotide sequence of the probe for detecting the Borrelia burgdorferi is shown as SEQID No. 23;
the nucleotide sequence of the probe for detecting the deer epidemic hemorrhagic fever virus is shown as SEQ ID NO. 24;
the nucleotide sequence of the probe for detecting the vesicular stomatitis virus is shown as SEQ ID NO. 25;
the nucleotide sequence of the probe for detecting the West Nile fever virus is shown as SEQID NO. 26;
the nucleotide sequence of the probe for detecting the coxiella burnetii is shown as SEQID NO. 27;
the nucleotide sequence of the probe for detecting rift valley fever virus is shown as SEQID NO. 28;
the nucleotide sequence of the probe for detecting the Ebola virus is shown as SEQID NO. 29;
the nucleotide sequence of the probe for detecting the Schmallenberg virus is shown as SEQ ID NO. 30;
the working procedure of the kit comprises the following steps:
(1) extracting nucleic acid of a sample to be detected;
(2) performing RT-PCR reaction on the sample RNA by using the 10 pairs of primers; in an RT-PCR reaction system, the concentration ratio of upstream primers to downstream primers of each primer in 10 pairs of primers is 1: 2; the RT-PCR reaction system 50 mu L comprises: one-step RT-PCR mix buffer 23. mu.L, RNA template 5.0. mu.L, 10 upstream primers 0.5. mu.L each, 10 downstream primers 1.0. mu.L each, and ddH without RNase2O is complemented to 50 mu L; the PCR reaction conditions are as follows: 30min at 50 ℃; 15min at 95 ℃; 35 cycles of 95 ℃ for 40s, 54 ℃ for 40s and 72 ℃ for 1 min; 10min at 72 ℃;
(3) carrying out molecular hybridization on a PCR product with a biotin label and a liquid chip containing the 10 probes, detecting a fluorescence value, and determining whether a sample contains African swine fever virus, bluetongue virus, borrelia burgdorferi, deer epizootic hemorrhagic fever virus, vesicular stomatitis virus, West Nile virus, Coxiella burnetii, rift valley fever virus, Ebola virus and/or Schmallenberg virus;
a50. mu.L hybridization system consisting of the PCR product and the liquid phase chip was configured as follows: 33 mu L of microsphere mixed solution, 15 mu L of TE Buffer with pH of 8.0 and 2 mu L of positive PCR product; the microsphere mixed solution is prepared by adding 1 mu L of each of 10 coupled microspheres to 330 mu L of 1 XTMAC hybridization buffer solution.
2. The use of claim 1, wherein the hybridization conditions are: denaturation at 95 ℃ for 5min, and hybridization at 50 ℃ for 30 min.
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