CN111073999B

CN111073999B - Visual chip for synchronously and jointly detecting six avian viruses

Info

Publication number: CN111073999B
Application number: CN201811233874.6A
Authority: CN
Inventors: 文翼平; 文心田; 向华
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2018-10-22
Filing date: 2018-10-22
Publication date: 2022-08-09
Anticipated expiration: 2038-10-22
Also published as: CN111073999A

Abstract

The invention provides a visual gold-labeled silver-stained gene chip which can simultaneously detect at least one virus of avian leukemia virus, Marek's disease virus, infectious bursal disease virus, Newcastle disease virus, avian influenza virus or infectious laryngotracheitis virus. The invention also provides a detection method of avian leukemia virus, Marek's disease virus, infectious bursal disease virus, Newcastle disease virus, avian influenza virus or infectious laryngotracheitis virus, which is realized by utilizing the chip. The method has the advantages of low cost, simple operation and higher flux, and has wide application prospect in the field of avian virus detection.

Description

Visual chip for synchronously and jointly detecting six avian viruses

Technical Field

The invention relates to the field of virus detection, in particular to visual chip detection of six avian viruses.

Background

Avian Leukemia (AL) is a collective term for a variety of neoplastic diseases in birds caused by viruses of the Avian C-type retrovirus group, mainly lymphocytic leukemia, and secondly erythroblastic leukemia and myeloblastic leukemia. Furthermore, it can cause myeloid tumor, connective tissue tumor, epithelial tumor, endothelial tumor, etc. Most tumors invade the hematopoietic system, and a few invade other tissues.

Marek's Disease (MD), also known as neurolymphomatosis (neurolymphomas), is a lymphoproliferative disease of chickens characterized by mononuclear cell infiltrates into peripheral nerves, gonads, irises, various internal organs, muscle and single or multiple tissue organs of the skin. The disease is an infectious neoplastic disease caused by cell-associated herpes virus, resulting in the formation of tumors in the above-mentioned organs and tissues. Sick chickens are usually emaciated and paralyzed in limbs, and are often killed acutely.

Infectious Bursal Disease (IBD) of chicken is also known as Gabor disease, and is an acute and highly Infectious disease caused by Infectious bursal disease virus. The disease is sudden in onset, short in course of disease, high in death rate and capable of causing immunosuppression of chicken bodies, and is still one of the main infectious diseases of the chicken raising industry at present.

Newcastle Disease (ND) is an acute, febrile, septic and highly contagious infectious disease of birds caused by newcastle disease virus. Characterized by high fever, dyspnea, diarrhea, neurological disturbance, mucosal and serosal bleeding. Has high morbidity and mortality, and is a main infectious disease harmful to poultry industry.

Avian Influenza (AI) is a complex condition caused by Influenza a viruses of the orthomyxoviridae family, highly pathogenic Avian Influenza can lead to 100% morbidity and mortality, virulent infectious diseases that are harmful to human and poultry health.

Infectious Laryngotracheitis (ILT) is an acute respiratory Infectious disease, and birds exhibit mental retardation, loss of appetite, drooping or lateral bending of the head, accumulation of small amounts of secretions in the eyes and nostrils, mouth opening, breathing, occasional spurting of bloody mucus or coagulated blood. Birds often die by asphyxiation due to excessive exudate and blood accumulation in the throat and trachea. At present, no effective treatment medicine exists.

The six poultry diseases are easily infected epidemic diseases of poultry, and in order to prevent the excessive breeding loss and even influence on human health, the main prevention and treatment method still detects corresponding pathogenic viruses and destroys hosts at present.

Common PCR, real-time fluorescent quantitative PCR and gene chip technology are common molecular detection means and are widely used for detecting poultry viruses. The sensitivity of common PCR is low, and generally only viruses with similar clinical symptoms can be identified, but different subtypes and subgroups of the same virus are difficult to identify; the specificity requirement of the real-time fluorescent quantitative PCR primer design is very high, and the high-throughput detection of samples can not be realized. The gene chip has the advantages of high flux, good specificity, strong sensitivity and the like.

However, no chip for simultaneously detecting six common avian viruses including avian leukemia virus, Marek's disease virus, infectious bursal disease virus, Newcastle disease virus, avian influenza virus and infectious laryngotracheitis virus is available at present.

At present, the traditional gene chip is usually adopted for detecting the avian viruses, and the technology needs to scan and read results by virtue of a fluorescence scanner which has the disadvantages of high cost, large size and complex operation. For example, patent CN102191339B discloses a chip for detecting several avian influenza subtypes, newcastle disease virus and egg drop syndrome virus; but because the fluorescence scanner is used, the fluorescence scanner has no universality in the field of avian virus detection.

The visualized chip for gold-labeled silver staining and color development can directly read the detection result by naked eyes without a fluorescence scanner, but the visualized chip for gold-labeled silver staining and color development is not reported for avian virus detection so far.

Disclosure of Invention

In order to solve the problems, the invention provides a group of avian virus detection probes, the nucleotide sequences of which are shown in SEQ ID NO. 1-7; the sequence of the avian leukemia virus probe is shown as SEQ ID NO.1, the sequence of the Marek's disease virus probe is shown as SEQ ID NO.2, the sequence of the chicken infectious bursal disease virus probe is shown as SEQ ID NO.3, the sequence of the Newcastle disease virus probe is shown as SEQ ID NO.4, the sequence of the avian influenza virus probe is shown as SEQ ID NO.5, and the sequence of the infectious laryngotracheitis virus probe is shown as SEQ ID NO. 6; the sequence of the positive control probe is shown as SEQ ID NO. 7; the 5' end of the probe is connected with an amino group.

The invention provides a group of PCR primers, the nucleotide sequence of which is shown as SEQ ID NO. 8-21; the sequence of the avian leukemia virus primer is shown as SEQ ID NO.8-9, the sequence of the Marek's disease virus primer is shown as SEQ ID NO.10-11, the sequence of the chicken infectious bursal disease virus primer is shown as SEQ ID NO.12-13, the sequence of the Newcastle disease virus primer is shown as SEQ ID NO.14-15, the sequence of the avian influenza virus primer is shown as SEQ ID NO.16-17, and the sequence of the infectious laryngotracheitis virus primer is shown as SEQ ID NO. 18-19; the sequence of the positive control primer is shown as SEQ ID NO. 20-21; the 5' end of the downstream primer is provided with a biotin label.

The invention provides a visual gene chip for detecting avian viruses, which comprises an aldehyde group modified matrix membrane and the avian virus detection probe; the substrate film is a silicon wafer or glass.

The invention also provides a preparation method of the chip, which comprises the following steps:

(1) designing an array of probe locations;

(2) diluting the oligonucleotide probe;

(3) using a chip spotting system to perform corresponding probe of the step (2) on the matrix film spot according to the array in the step (1), and drawing a frame along the probe array by using a marking pen on the back of the chip;

(4) ultraviolet crosslinking for 6-12 min, and hydrating overnight at 37 ℃;

(5) sealing with sealing liquid, cleaning, air drying, and storing at 4 deg.C.

Further, the confining liquid in the step (5) comprises: 0.4% BH ₄ Na and 20% -30% of ethanol.

The invention also provides a visual detection method capable of detecting the avian viruses, which adopts the chip for detection.

The detection method comprises the following steps:

A) DNA amplification: amplifying the sample DNA by using the primer of claim 4, and mixing the amplified products to form a sample mixed solution;

B) and (3) hybridization: performing high-temperature denaturation on the sample mixed solution for 5 minutes, immediately performing ice bath for 3 minutes, dropwise adding the sample mixed solution into a chip array area containing a fence for fixation, standing for 60-150 minutes at the temperature of 35-55 ℃, washing with ultrapure water, and drying at room temperature;

C) and (3) incubation: adding 1.6-8 mug/mL Nanogold-Streptavidin Streptavidin solution into the chip array area, incubating for 30min at 37 ℃, washing with ultrapure water, and airing at room temperature;

D) silver staining: adding silver staining reagent in dark place, standing for 3-9min, cleaning with ultrapure water, and naturally drying;

E) interpretation: and (4) judging the detection to be positive if black spots appear in the corresponding probe areas.

Preferably, the standing temperature of step B) is 40 ℃ and the standing temperature is 120 min;

preferably, the Nanogold-Streptavidin Streptavidin solution concentration of step C) is 4. mu.g/mL.

Preferably, the standing time of step D) is 5 min.

The kit can synchronously detect six common avian viruses including avian leukemia virus, Marek's disease virus, infectious bursal disease virus, Newcastle disease virus, avian influenza virus and infectious laryngotracheitis virus, and has high specificity. Compared with a PCR/RT-PCR method, the specificity coincidence rate is more than 97.7 percent.

In addition, the sensitivity of the invention is very high, and the minimum detection concentration is 1 pg/mu l.

The method can be used for synchronously and co-detecting 6 common avian viruses accurately, sensitively and at low cost, and has wide application prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows a design of a gene chip matrix.

FIG. 2 shows the results of target gene identification: m: 2000bp DNA Ladder; 1: a lambda localization gene; 2: ALV-env; 3: MDV-meq; 4: IBDV-VP ₂ (ii) a 5: NDV-F; 6: ILTV-TK; 7: AIV-NP; n: and (5) negative control.

FIG. 3 is a detection map of lambda localizing genes.

FIG. 4 is a detection diagram of the full probe.

Fig. 5 shows the results of the optimization of the number of times of sample injection: a: spraying the sample once, B: sample spraying twice, C: and spraying samples for three times.

FIG. 6 shows the hybridization temperature optimization results of the visual gene chip: a: 35 ℃; b: 40 ℃; c: 45 ℃; d: 50 ℃; e: at 55 ℃.

FIG. 7 shows the result of analysis of hybridization temperature.

FIG. 8 shows the optimized hybridization time results of the visual gene chip: a: 1 h; b: 1.5 h; c: 2 h; d: 2.5 h.

FIG. 9 shows the result of analysis of hybridization time.

FIG. 10 shows the results of optimizing the concentration of Nanogold-Streptavidin solution on the visual gene chip: a: 8 mu g/mL; b: 4 mu g/mL; c: 2.67. mu.g/mL; d: 2 mu g/mL; e: 1.6. mu.g/mL.

FIG. 11 shows the silver staining time optimization results: a: 3 min; b: 5 min; c: 7 min; d: and 9 min.

FIG. 12 is a gray scale histogram of silver staining time.

FIG. 13 shows the results of the visual gene chip specificity test: a: ALV; b: MDV; c: IBDV; d: NDV; e: an AIV; f: an ILTV; g: IBV.

FIG. 14 shows the results of the visual gene chip sensitivity test: a: 1 ng/. mu.l; b: 100pg/μ l; c: 10 pg/. mu.l; d: 1 pg/. mu.l; e: 100 fg/. mu.l.

FIG. 15 shows the visualized gene chip stability test results (at room temperature): a: 30 days; b: 60 days; c: and 75 days.

FIG. 16 shows the results of the gene chip stability test (4 ℃ C.): a: 30 days; b: 60 days; c: 75 days; d: for 90 days.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments in the form of examples and experimental examples. However, it should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples and experimental examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Abbreviations:

ALV, avian leukemia virus;

MDV, marek's disease virus;

IBDV, infectious bursal disease virus of chickens;

NDV, newcastle disease virus;

AIV, avian influenza virus;

ILTV, infectious laryngotracheitis virus;

IBV, infectious bronchitis virus.

The main reagents and main instruments used in the examples and experimental examples are as follows:

1) the main reagents are as follows:

tiangen (Beijing) Corp: tissue genome DNA extraction kit, total RNA extraction kit and DNA product purification kit

Omega company, usa: DNA plasmid extraction kit

Beijing Boao BioLimited: crystal core sample application liquid, aldehyde-based substrate and 12-sample chip fence

Sigma, USA: silver staining reagent: silver buffer A and Silver buffer B

Nanoprobes, USA: Nanogold-Streptavidin Streptavidin

Sealing liquid: according to 0.5% BH ₄ Na, 25% Ethanol, 0.75 × PBS.

Baoybio (Dalian) Co., Ltd: PrimeScript RT Reagent Kit

2) The main apparatus is as follows:

sichuan crown gas Co., Ltd: high-purity nitrogen cylinder

Beijing Capitalbio biological Co., Ltd: chip hybridization box and hybridization related product

Beijing Boo ao Biopsis:

SmartArrayer ^TM 16, Capitalbio Corporation gene chip spotting system

Thermo corporation, usa: molecular hybridization instrument

Chengdu Wanke Co., Ltd: operating forceps, tweezers, ophthalmic scissors and alcohol burner

Example 1 preparation of visual Gene chip

1. Probe synthesis

Probes were synthesized according to Table 1, with 1 amino group at each 5' end of the probe.

TABLE 1 Probe sequence Listing

2. Array design

Designing gene chip arrays according to the size of the aldehyde-based substrate and the number of probes, wherein each chip has 3 repeated arrays, and each repeated array is designed with 4 rows of probe genes. The position of the probe of each virus target gene on the substrate is as follows: the first three of the first rows are negative controls, the last three of the first rows are positive controls (lambda localizing gene), the first three of the second rows are ALV, the last three of the second rows are MDV, the first three of the third rows are IBDV, the last three of the third rows are NDV, the first three of the fourth rows are AIV, and the last three of the fourth rows are ILTV, as shown in FIG. 1.

3 dilution of visual Gene chip Probe

Spotting buffer and ddH ₂ O is mixed uniformly in a ratio of 1: 4 to dilute the synthesized oligonucleotide probe and adjust the final concentration to 50. mu. mol/L. The diluted oligonucleotide probe mixture (80. mu.L) was pipetted gently into the corresponding wells of a 384-well plate, each well representing a target gene probe dilution, and the wells of the two target genes were separated by two wells to avoid contamination between the target gene mixtures (note that the probe mixtures could not have air bubbles in the wells). The 384 well plate was placed in the appropriate position on the spotting instrument. Meanwhile, the aldehyde glass slide is placed on a module of a sample applicator, and the sample applicator is corrected once before sampling.

4 correction of visual gene chip sample applicator

The power supply of the computer and the sample applicator is turned on, and the sample application needle is arranged in the corresponding position of the sample applicator. And opening a chip Personal array 16 operating software on a computer, entering a setting interface after self-checking is finished, controlling the ambient humidity in the spotting instrument to be more than 50%, and respectively performing sample spraying preparation, slide setting, dot matrix setting, sample setting and cleaning setting operations. In the preparation of the sample ejection, the cleaning position, the draining position, the first slide position, the pre-ejection position, the sample well plate a24 well position, and the emptying position were corrected, respectively. OX in slide setup: 5.5mm, OY: 7mm, initial slide 1, number of sprayed-on slides set according to the actual number of put-in. Dot pitch X in the dot matrix setup: 1800 μm, Y: 2200 μm; number of needle array points X: 6, Y: 4; counting the number of repeated samples: 3; inter-array interval X: 1mm, Y: 12.5 mm; the array number X: 1, Y: 3; array repeat: is. Cleaning in the cleaning setting: 5 times, emptying: 4 times, pumping to dry: the cycle was repeated 4 times. And opening the nitrogen, and starting to perform sample injection, wherein the sample injection times are 1.

5 spraying and sealing fixation of visual gene chip

And (3) carrying out non-contact sample spraying according to the designed detection array arrangement (as shown in figure 1). After the sample spraying is finished, the chip is subjected to ultraviolet crosslinking for 10min along a probe array picture frame by using a marking pen on the back surface of the chip, and the chip is placed in a wet box containing purified water and hydrated at 37 ℃ overnight. Subsequently, the chip multi-sample pens were capped on the chip. 0.5% NaBH at 37 deg.C ₄ Sealing solution is added into the chip array area containing the fence fixation, and sealing is carried out for 30 min. The chip was taken out, and extracted and cleaned with ultrapure water for 5 times. Naturally drying, and storing at 4 deg.C for use.

Example 2 visual Gene chip detection

1. Material

Target gene recombinant plasmid: T/AIV-NP, T/DNV-F, T/ILTV-TK, T/IBV-N; lambda targeting gene recombinant plasmids; e.coli DH5 α; MDV, IBDV virus tissue fluid; DNA nucleic acid of ALV.

2 method

2.1 design and Synthesis of primers

The gene sequences of ALV, MDV and IBDV strains recorded by NCBI are compared and analyzed by Megalign of DNASTAR software, 3 pairs of primers (env gene of ALV, meq gene of MDV and VP2 gene of IBDV) are designed by Primer5.0, the amplification length of a target gene is between 200 and 800bp, Tm is (55 +/-5) DEG C, the primers are synthesized by Beijing Populaceae New technology Limited company (see table 2), and the 5' end of the downstream primer of the target gene is modified by biotin.

TABLE 2 primers for the target genes of ALV, MDV and IBDV

2.2 amplification and identification of the target genes ALV-env, MDV-meq, IBDV-VP2

Extracting total RNA of virus strain IBDV and reversely transcribing the total RNA into cDNA; and (3) detecting and identifying the DNA of ALV and MDV by gel electrophoresis after PCR amplification according to primers designed for ALV-env, MDV-meq and IBDV-VP2 target genes. And after the gel electrophoresis identification result is correct, recovering the target gene by using a gel recovery kit. The gel recovery product was ligated with pMD19-T Simple vector, ligated for 10h at 16 ℃ and 10. mu.l of the ligation product was transformed into 50. mu.l of DH 5. alpha. competent cells (note that repeated freeze-thawing of competent cells was prohibited). The transformation solution was aspirated through an aluminum cap, plated on an Amp-resistant LB plate, and cultured overnight. And selecting the suspected positive monoclonal on the culture plate, putting the suspected positive monoclonal into 20 mu l of LB liquid culture medium containing Amp resistance, sucking 1 mu l of the suspected positive monoclonal for PCR amplification identification, and storing the remaining 19 mu l of the suspected positive monoclonal in a refrigerator at 4 ℃ for later use. After the PCR amplification identification sequence size is correct, 19 mul of bacterial liquid is subjected to amplification culture, plasmids are extracted and sent to Shanghai to carry out sequencing, the sequencing result is compared with the reference strain recorded in NCBI, and the sequencing result is shown in Table 3, which shows that the sequence is reliable.

TABLE 3 ALV-env, MDV-meq and IBDV-VP ₂ Identification result of target Gene

2.3 Resuscitation and identification of target genes

Recovering the recombinant plasmid bacteria frozen at-70 deg.C, and extracting recombinant plasmid ALV-env, MDV-meq, IBDV-VP2, AIV-NP, NDV-F, ILTV-TK, IBV-N, and lambda localization genes. The extracted plasmid DNA was then diluted 50-fold and the diluted DNA was used as a template for PCR amplification. The localized gene was amplified by PCR using phage lambda DNA as template.

Reaction procedure: 1)95 ℃ for 5 min; 2) at 95 ℃ for 30 s; (wherein the temperature of the targeting gene, AIV-NP, NDV-F, ILTV-TK is 56.5 ℃, ALV-env, MDV-meq, IBDV-VP ₂ At a temperature of 52 ℃) for 30 s; 30s at 72 ℃ for 30 cycles; 3)72 ℃ for 8 min; 4)12 ℃ and forever. 8 μ L of the amplification product was identified by gel electrophoresis (FIG. 2). Primers of the target genes of the AIV-NP, DNV-F, ILTV-TK, IBV-N and lambda positioning gene recombinant plasmids constructed and stored in the laboratory of Sichuan university of agriculture are synthesized by Beijing Okagaku (see Table 4), and the 5' end of the downstream primer of the target gene is modified by biotin. Among them, IBV was used as a specific test in Experimental example 1.

TABLE 4 primers for target genes of AIV, NDV, ILTV, IBV and phages

Note: f is an upstream primer; r is a downstream primer;

2.4 visual chip detection

The detection steps mainly comprise hybridization, incubation of Nanogold-Streptavidin Streptavidin solution, development of silver staining reagent and judgment of visual gene chip results. And (3) hybridization: the step 2.3 amplification marker comprises ALV-env, MDV-meq and IBDV-VP ₂ The target gene PCR products of the AIV-NP, NDV-F, ILTV-TK and the lambda localization gene are fully and uniformly mixed, and are denatured in 100 ℃ water bath for 5min and immediately ice-bathed for 3min, so that the double-strand renaturation of DNA is prevented. Adding the mixed solution of the PCR products into a chip array area containing a fence for fixation, hybridizing for 2h at 40 ℃, extracting and cleaning with ultrapure water for five times after hybridization, and naturally drying at room temperature after cleaning; incubation of Nanogold-Streptavidin Streptavidin: the Nanogold-Streptavidin solution with the concentration of 4 mug/mL is slowly added to the corresponding chipArray area, incubated at 37 ℃ for 30 min. After the incubation is finished, extracting and cleaning with ultrapure water for five times, and naturally airing at room temperature after cleaning; developing with silver staining reagent: and (3) under the dark condition, uniformly mixing the Silver dye reagent of Silver buffer A and Silver buffer B in equal volume, adding 200 mu l of mixed solution into the chip array area, and standing for 5 minutes.

3. Results

3.1 detection of Positive localized Gene

And (3) carrying out target gene amplification and labeling by taking the lambda positioning gene as a template, hybridizing a lambda DNA amplification product with a chip containing 6 avian virus disease probes, carrying out silver staining for color development, and observing the test result by naked eyes. The results showed that hybridization spots were clearly visible for the positive control lambda localization gene, and no hybridization spots were visible for the negative control and other probe spots (FIG. 3). The aldehyde group substrate and the positive positioning gene prepared by the research have reliable quality.

3.2 detection of the Total Gene of the Co-detection chip

All probe sites of 6 probe genes of the six avian viral diseases can see obvious hybridization spots, positive control spots are obvious, and negative control spots are free of visible hybridization spots, so that the quality of the prepared visual co-detection chip and the selected probe genes is reliable (figure 4).

Example 3 visual Gene chip number of puffs optimization

1. Method of producing a composite material

The oligonucleotide probe solution at a final concentration of 50 pmol/L was added to the corresponding well of a 384-well sample plate as ddH ₂ O as a negative control. And (3) spraying samples on 3 aldehyde-based substrates for 1 time, 2 times and 3 times in sequence by using a chip sample applicator according to the step 2.5.4, preparing chips with different sample spraying times, and sealing and fixing the chips. Subsequently, the results were observed following the procedure for visualization of chip detection in example 2.

2. Results

The result shows that the spot signals are not obviously different when one or two samples are sprayed, but when the third sample is sprayed, the hybridization signals are connected together due to the displacement among the probes, and the judgment of the final hybridization result is influenced. And as the number of sample injections increases, the test cost and time increase. Therefore, a chip was prepared by selecting one-time sampling (see FIG. 5).

Example 4 visual Gene chip hybridization temperature optimization

1. Method of producing a composite material

5 chips prepared under the same conditions were taken, 200. mu.L of the mixed solution of the target gene products was added to the array region of the chip, fixed with a fence, and then placed in a hybridization cassette. Respectively hybridizing the amplified and labeled target gene product mixed solution with the chip for 120min at the temperature of 35 ℃, 40 ℃, 45 ℃, 50 ℃ and 55 ℃. Taking out the chip, washing with ultrapure water for 5 times, and air drying the chip in natural environment. Adding 4 mug/ml Nanogold-Streptavidin solution to the corresponding position of the pen containing the probe, incubating for 30min at 37 ℃, finally staining for 5min by using silver staining reagent, and judging the optimal condition of the hybridization temperature of the gene chip.

2. Results

The eye results show (see fig. 6): when the hybridization temperature is 35 ℃, the hybridization signal of the corresponding position of the chip is weak, the hybridization signal is enhanced along with the increase of the temperature, and when the temperature is 40 ℃ and 45 ℃, the obvious hybridization signal can be seen at the corresponding position of the chip. Whereas, when the temperature is higher than 50 ℃, the hybridization signal of the individual target gene starts to decrease, and then more target gene hybridization signals are weak. Gray-value data analysis display (see fig. 7): when the temperature is lower, the hybridization signal is gradually increased along with the increase of the temperature, and when the temperature is increased to a certain degree, the value of the hybridization signal is not increased any more and then is gradually reduced; when the temperature is higher than 45 ℃, the change of the background value is not obvious; the difference between the hybridization signal value and the background value is maximal at a temperature of 40 ℃. Therefore, 40 ℃ was selected as the optimal hybridization temperature for this experiment.

Example 5 optimization of visual Gene chip hybridization time

1. Method of producing a composite material

Similarly, four gene chips prepared in the same batch were taken, 200. mu.L of the mixed solution of the target gene products was added to the array region of the chip, and the chip was fixed with a fence and then placed in a hybridization cassette. The mixture of the amplified and labeled target gene products and the chip are respectively hybridized for 60min, 90min, 120min and 150min at the temperature of 40 ℃. Taking out the chip, washing with ultrapure water for 5 times, and naturally drying. Add 4. mu.g/ml Nanogold-Streptavidin solution to the pen containing probe corresponding position, 37 degrees C were incubated for 30min, finally using silver staining reagent staining for 5 minutes. Determining the optimal hybridization time of the gene chip.

2. Results

The eye results show (see fig. 8): when the hybridization time is 1h, the hybridization signal at the corresponding position of the chip is weak, the hybridization signal is enhanced along with the prolonging of the hybridization time, and when the hybridization time reaches 2h, the hybridization signal intensity effect is best. Subsequently, as the hybridization time is continuously prolonged, the hybridization signal value and the background value of the gene chip are simultaneously enhanced, and the enhancement of the signal value is far lower than the enhancement speed of the background value by the visual observation. The gray-scale data analysis results (see fig. 9) show that the hybridization signal value and the background value are both increased with the increase of the hybridization time, and the difference between the hybridization signal value and the background value is the largest when the hybridization time is 120 min. Therefore, 2h was chosen as the optimal hybridization time for this experiment.

Example 6 optimization of visualization Gene chip Nanogold-Streptavidin Streptavidin solution

1. Method of producing a composite material

Using 5 simultaneous prepared gene chips, 200. mu.L of the target gene product mixture was added to the array region of the chip, and the chip was fixed with a pen and placed in a hybridization cassette. The mixture of the amplified and labeled target gene products was hybridized with the chip at 40 ℃ for 120 min. Taking out the chip, washing with ultrapure water for 5 times, and naturally drying. Then, the corresponding array region of the chip was added with Nanogold-Streptavidin solution at a concentration of 8. mu.g/mL, 4. mu.g/mL, 2.67. mu.g/mL, 2. mu.g/mL, 1.6. mu.g/mL, respectively, incubated at 37 ℃ for 30min, and finally stained with silver staining reagent for 5 min. The optimum concentration of Nanogold-Streptavidin solution applied to the gene chip is determined.

2. Results

The results show (see fig. 10): as the concentration of Nanogold-Streptavidin Streptavidin solution decreased, the hybridization signal also gradually decreased. Binding reagent cost Nanogold-Streptavidin solution was chosen at the optimal concentration at a concentration of 4. mu.g/mL.

Example 7 visual Gene chip silver staining time optimization

1. Method of producing a composite material

4 identical gene chips were taken, 200. mu.L of the mixed solution of the target gene products was added to the array region of the chip, and the chip was fixed with a fence and then placed in a hybridization cassette. The mixture of the amplified and labeled target gene products was hybridized with the chip at 40 ℃ for 120 min. The chip was taken out and washed with ultrapure water 5 times. After air-drying, incubation with 4. mu.g/ml Nanogold-Streptavidin solution was used. Adding silver staining reagent in dark condition, and performing silver staining for 3min, 5min, 7min and 9 min. And after the color development is finished, cleaning the chip by using ultrapure water, naturally drying, and observing test results by naked eyes to determine the optimal silver staining time of the visualized gene chip.

2. Results

The results showed (see FIGS. 2-11) that the hybridization signal value and background value of the gene chip increased with the increase of the silver staining time. The results of grey scale data analysis showed that after 5min of silver staining, the hybridization signal values did not increase significantly and the background values increased significantly as the silver staining time was increased (see FIGS. 2-12). Therefore, 5min was selected as the optimal silver staining time.

Experimental example 1 specificity test of visual Gene chip

1. Method of producing a composite material

1.1 extraction of target Gene recombinant plasmid

The target gene recombinant plasmids T/ALV-env, T/MDV-meq, T/IBDV-VP2, T/DNV-F, T/AIV-NP, T/ILTV-TK, T/IBV-N, lambda targeting gene were obtained in the same manner as in example 2.

Extracting target gene recombinant plasmids T/ALV-env, T/MDV-meq, T/IBDV-VP2, T/DNV-F, T/AIV-NP, T/ILTV-TK, T/IBV-N and lambda localization genes according to the instruction of a DNA plasmid extraction kit, adjusting the initial concentration of the extracted plasmids to 1 ng/mu l, and sequentially carrying out 10 steps ¹ -10 ⁵ And (5) diluting by times.

1.2 amplification labeling of target Gene

PCR amplification was performed using the above plasmid as a template, according to the primers shown in Table 2 and Table 4 in example 2.

The amplification system is as follows:

2×Taq PCR Master mix	25μL
		upstream primer	2μL
Biotin-labeled downstream primer	2μL
		Plasmid template	2μL
Deionized water	19μL
		Total volume	50μL

The amplification conditions were: 1) pre-denaturation at 95 ℃ for 5 min; 2) denaturation at 95 ℃ for 30s, annealing (where the temperatures of lambda targeting gene, AIV-NP, NDV-F, ILTV-TK, and IBV-N are 56.5 ℃ and ALV-env, MDV-meq, and IBDV-VP2 are 52 ℃) for 30s, and extension at 72 ℃ for 45s for 30 cycles; 3) finally, the mixture is extended for 10min at 72 ℃ and stored at 12 ℃.

1.3 preparation of visual chip for common inspection

Chips were prepared according to the method of example 1.

1.4 specificity test

Mixing ALV-env, MDV-meq, IBDV-VP ₂ NDV-F, AIV-NP and ILTV-TK, and the single-stranded target genes amplified by the NDV-F, AIV-NP and ILTV-TK are separately hybridized and developed with a chip, and the hybridization result is observed visually. Meanwhile, IBV-N gene amplification products stored in the laboratory are hybridized with a visual chip, and the specificity of the gene chip is evaluated by observing results.

2. As a result, the

When ALV-env, MDV-meq, IBDV-VP2, NDV-F, AIV-NP and ILTV-TK were hybridized with the visual chip alone, only macroscopic hybridization spots appeared at the corresponding positions of the chip, and there was no non-specific hybridization between the individual probes. After the IBV-N gene amplification product is hybridized with the visual chip, no hybridization signal spot is visible to the naked eye at the corresponding position of the chip, and the hybridization result is negative (FIG. 13). Thus, the gene chip constructed in this test has high specificity.

Experimental example 2 sensitivity test of visual Gene chip

1. Method of producing a composite material

1.1 extraction of target Gene recombinant plasmid

The same as in experimental example 1.

1.2 amplification labeling of target Gene

The same as in experimental example 1.

1.3 preparation of visual chip for common inspection

The same as in experimental example 1.

1.4 sensitivity test

Using dd H ₂ O the extracted target Gene plasmids (initial concentration 1 ng/. mu.L) were sequentially subjected to 10 ¹ -10 ⁵ Diluting, PCR amplifying and marking, and mixing ALV-env, MDV-meq and IBDV-VP ₂ The single-stranded target gene products amplified by NDV-F, AIV-NP and ILTV-TK are mixed, hybridized and developed with a chip, and the hybridization result is observed visually to evaluate the sensitivity of the gene chip.

2. Results

When the concentration of the template is 1 ng/mu l, 100 pg/mu l and 10 pg/mu l, obvious hybridization signals can be seen at the corresponding positions of the chip; when the template concentration is 1 pg/. mu.l, the hybridization signal of a part of the target gene is continuously decreased as the template concentration is decreased. When the concentration of the template was 100 fg/. mu.l, no hybridization signal was observed with the naked eye (FIG. 14). The lowest detection concentration of the detection chip is 1 pg/. mu.l.

Experimental example 3 stability test of visual Gene chip

1. Method of producing a composite material

1.1 extraction of target Gene recombinant plasmid

The same as in experimental example 1.

1.2 amplification labeling of target Gene

The same as in experimental example 1.

1.3 preparation of visual chip for common inspection

The same as in experimental example 1.

1.4 stability test

The gene chip is sealed and stored at normal temperature and 4 deg.c separately. One chip was randomly picked up at 30 days, 60 days, 75 days, and 90 days, respectively. Mixing ALV-env, MDV-meq, IBDV-VP ₂ The single-stranded target gene products amplified by NDV-F, AIV-NP and ILTV-TK were mixed, hybridization and coloration were performed with the chip, and the visual results were visualized by silver staining to evaluate the stability of the chip.

2. As a result, the

The chip stored at normal temperature and 4 ℃ within 60 days has no obvious influence on hybridization signals, and the detection signal points are obvious. As the storage time of the chip is prolonged, the hybridization signal spots become blurred and the signal spots of individual target genes disappear at 75 days of the chip under the normal temperature condition. On the other hand, in the chip at 4 ℃ for 90 days, the hybridization signal spots appeared blurred, and the signal spots of the individual target genes disappeared (FIGS. 15 and 16). The visual chip established in the research can be stored for 75 days at 4 ℃ and 60 days at normal temperature.

Experimental example 4 clinical application of gene chip for visual co-detection of six avian viruses

1. Biological material

The tissues of laryngotracheae, lungs, spleens, livers, kidneys and bursa of fabricius of Sichuan Anyue, Pengzhou, Luzhou, Guangyuan, Yuong, Leshan, Guanhan, Wenjiang, Chongqing, Shandong and other regions suspected to be avian leukemia, Marek's disease, infectious bursal disease virus, avian influenza, Newcastle disease and infectious laryngotracheitis of chickens, spotted-foot partridge, white feather broilers and black-bone chickens.

2 method

2.1 extraction of clinical sample nucleic acid and PCR amplification labeling

The tissues of laryngotracheae, lung, spleen, bursa of fabricius, liver and kidney of suspected sick chicken are aseptically collected and cut into pieces by ophthalmic scissors. Total RNA and DNA extraction of the genome was performed with reference to the instructions. And performing cDNA reverse transcription by using the extracted RNA as a template. The reverse transcription procedure was as follows:

the reaction program was 37 deg.C, 15min, 85 deg.C, 5sec, 4 deg.C storage. Meanwhile, the reverse transcription product cDNA and the extracted DNA are placed at-20 ℃ and stored for later use.

2.2 extraction of Positive plasmids

Positive plasmids for ALV-env, MDV-meq, IBDV-VP2, AIV-NP, DNV-F, ILTV-TK were extracted as described in section 2.2 of example 2.

2.3 preparation of visual Gene chip

Visual gene chips required for this experiment were prepared in the same batch as in example 2

ALV-MDV-IBDV-NDV-AIV-ILTV joint co-detection chip.

2.4 comparison of PCR/RT-PCR and visual Gene chip detection of clinical samples

(1) PCR/RT-PCR detection of six avian viral diseases

Total RNA and DNA extraction of the genome was performed on 155 clinical samples using RNA and DNA extraction kits, the extracted DNA was temporarily stored at-20 ℃ and RNA was inverted to cDNA for future use. Taking cDNA/DNA as a template, carrying out PCR amplification on the ALV-env, MDV-meq, IBDV-VP2, AIV-NP and DNV-F, ILTV-TK target genes, and taking the PCR amplification of the positive plasmid of the corresponding target gene as a positive control. Primer reference example 2, PCR reaction procedure was as follows:

2×Taq PCR Master mix	7.5μL
		ddH2O	4.5μL
downstream primer	1μL
		cDNA template	1μL
Downstream primer	1μL
		Total volume	15μL

Amplification conditions: the first step is as follows: pre-denaturation at 95 ℃ for 5 min;

the second step is that: denaturation at 95 ℃ for 30s, annealing (wherein the temperature of ALV-env, MDV-meq, IBDV-VP2 is 52 ℃ and the temperature of AIV-NP, NDV-F, ILTV-TK is 56.5 ℃) for 30s, extension at 72 ℃ for 45s, and 30 cycles;

the third step: extending for 10min at 72 ℃, and storing at 12 ℃. And carrying out 1% agarose gel electrophoresis identification on the amplification product.

(2) Visual gene chip for detecting six kinds of avian virus diseases

And carrying out PCR amplification marking on the target genes of ALV-env, MDV-meq, IBDV-VP2, DNV-F, AIV-NP and ILTV-TK by taking cDNA/DNA of the same batch of pathological materials as a template. The reaction conditions were the same as in section (1) of 2.4.

155 clinical specimens were tested using the same batch of ALV-MDV-IBDV-NDV-AIV-ILTV combined consensus chip prepared in chapter II, according to the method described in section 2.4 of example 2. The clinical application of the visual detection chip is preliminarily evaluated. The single-chain target gene marked by biotin is hybridized with the chip, the result of the eye-observation detection is stained by gold-marked silver, and the signal value and the background value of the chip are subjected to gray scanning analysis so as to evaluate the clinical application of the visual detection chip.

(3) Comparison of results coincidence rates of two detection methods

Taking the detection result of the RT-PCR method as a reference, if the detection result of the RT-PCR method is positive, the visual chip is also positive, and the detection is called true positive; if the former is positive, the detection result of the gene chip is negative and is called false negative; if the detection result of the former is negative and the chip is positive, the detection result is called false positive; the former is assumed to be negative, and the chip detection is negative and is called true negative. Statistical analysis of the sample test results was performed using SPSS 22.0 software: when the Kappa value is less than or equal to 0.40, the consistency of the two detection methods is poor; when the Kappa value is more than 0.40 and less than or equal to 0.80, the two detection methods have higher consistency; when the Kappa value is greater than 0.80, it indicates that the two detection methods have excellent consistency; when the Kappa value is 1.00, it indicates that the two detection methods are completely identical.

3. Results

3.1PCR/RT-PCR results for detecting six avian viral diseases

The PCR/RT-PCR results showed (Table 5-1): the positive detection rates of ALV, MDV, IBDV, NDV, AIV, and ILTV were 7.7%, 1.2%, 4.5%, 12.9%, and 0%, respectively, with 3.8% for mixed infections ALV and AIV, 1.9% for NDV and AIV, 0.6% for ALV, MDV, and IBDV, 0.6% for ALV, AIV, and IBDV, and 1.2% for ALV, AIV, and NDV.

3.2 visual Gene chip results of detecting six avian viruses

Visual gene chip assay results show (Table 5-1): the positive detection rates of ALV, MDV, IBDV, NDV, AIV, and ILTV were 7%, 1.2%, 0.6%, 5.8%, 14.8%, and 0%, respectively, with a mixed infection of ALV and AIV of 4.5%, NDV and AIV of 2.5%, ALV, MDV, and IBDV of 0.6%, and ALV, AIV, and NDV of 1.2%.

Table 5-1155 samples results of six avian viral disease detection

Table 5-1 The detection results of 155 samples for detecting six kinds ofpoultry virus diseases

Note: percent of agreement (%) - (number of true positive detection + number of true negative detection)/n.times.100%

Note：Coincidence rate＝(True-positive number+True-negtive number)/n×100％

3.3 percent of coincidence of two methods for detecting six avian viral diseases

The coincidence rate test result shows that: compared with PCR/RT-PCR, the sensitivity of visual chip detection is higher than 91.6%, the specificity is higher than 97.7%, the Kappa value is larger than 0.80, and some even reach 1 (except IBDV pathogen detection, the sensitivity is 50%, the Kappa value is less than or equal to 0.40). Indicating that the two detection methods have relatively good consistency (see Table 5-2). The six avian virus gene chips constructed can be used for preliminary detection application of clinical samples.

TABLE 5-2 evaluation of the practicability of the visual chip for detecting six avian viruses

Table 5-2 The practicability of the visual microarray for detecting six kinds of poultry virus diseases

Note: chip-specific calculation mode: the number of true negative detections (the number of false positive detections + the number of true negative detections) is 100%

Chip sensitivity calculation mode: the number of true positive detections (the number of false negative detections + the number of true positive detections) is 100%

The experiments show that the invention not only has better specificity, sensitivity and stability, but also can approach the gold standard-RT-PCR test result of molecular detection. The invention has good application prospect in the field of avian virus detection by combining the characteristics of simple operation, higher flux and lower cost.

SEQUENCE LISTING

<110> Sichuan university of agriculture

<120> visual chip for synchronously and co-detecting six avian viruses

<130> GY151-18P1528

<160> 21

<170> PatentIn version 3.5

<210> 1

<211> 47

<212> DNA

<213> ALV-P(artificial sequence)

<400> 1

tttttttttt tttttggatt tctgcctctc tacacagtca gccacct 47

<210> 2

<211> 43

<212> DNA

<213> MDV-P(artificial sequence)

<400> 2

tttttttttt tttttatgga gtttgtctac atagtccgtc tgc 43

<210> 3

<211> 49

<212> DNA

<213> IBDV-P(artificial sequence)

<400> 3

tttttttttt ttttttgaac tagcaaagaa cctggtcaca gaatacggc 49

<210> 4

<211> 45

<212> DNA

<213> NDV-P(artificial sequence)

<400> 4

tttttttttt tttttcaagc caaacaaaat gctgccaaca tcctc 45

<210> 5

<211> 46

<212> DNA

<213> AIV-P(artificial sequence)

<400> 5

tttttttttt tttttccctc tgttggttgg tgtttcctcc gcttct 46

<210> 6

<211> 48

<212> DNA

<213> ILTV-P(artificial sequence)

<400> 6

tttttttttt tttttctgcc tcatgaatac taccacctac ctccaacg 48

<210> 7

<211> 40

<212> DNA

<213> Positive control (artificial sequence)

<400> 7

tttttttttt tttttgattt ggagggcagt tgcggtcgtg 40

<210> 8

<211> 20

<212> DNA

<213> ALV-R(artificial sequence)

<400> 8

ggatgaggtg actaagaaag 20

<210> 9

<211> 19

<212> DNA

<213> ALV-F(artificial sequence)

<400> 9

gggaggtggc tgactgtgt 19

<210> 10

<211> 18

<212> DNA

<213> MDV-R(artificial sequence)

<400> 10

cagggagcag acggacta 18

<210> 11

<211> 18

<212> DNA

<213> MDV-F(artificial sequence)

<400> 11

gagggcagaa gagggaat 18

<210> 12

<211> 22

<212> DNA

<213> IBDV-R(artificial sequence)

<400> 12

ggcccagagt ctacaccata ac 22

<210> 13

<211> 21

<212> DNA

<213> IBDV-F(artificial sequence)

<400> 13

ccggattatg tctttgaagc c 21

<210> 14

<211> 18

<212> DNA

<213> AIV-R(artificial sequence)

<400> 14

ccagaagcgg aggaaaca 18

<210> 15

<211> 18

<212> DNA

<213> AIV-F(artificial sequence)

<400> 15

gtcaaaggaa ggcacgat 18

<210> 16

<211> 22

<212> DNA

<213> NDV-R(artificial sequence)

<400> 16

caagaaccca gcacctatga tg 22

<210> 17

<211> 19

<212> DNA

<213> NDV-F(artificial sequence)

<400> 17

gtcggaggat gttggcagc 19

<210> 18

<211> 23

<212> DNA

<213> ILTV-R(artificial sequence)

<400> 18

tcctcgtaga taggcaccca ctc 23

<210> 19

<211> 27

<212> DNA

<213> ILTV-F(artificial sequence)

<400> 19

tacgttggag gtaggtggta gtattca 27

<210> 20

<211> 21

<212> DNA

<213> λ-R(artificial sequence)

<400> 20

aaagcgacgc aatgaggcac t 21

<210> 21

<211> 19

<212> DNA

<213> λ-F(artificial sequence)

<400> 21

gttccacgac cgcaactgc 19

Claims

1. A group of primers and probe groups for detecting avian viruses is characterized in that: the nucleotide sequence of the probe is shown in SEQ ID NO. 1-7;

the 5' end of the probe is connected with an amino;

the nucleotide sequence of the primer is shown as SEQ ID NO. 8-21;

wherein, the 5' end of the downstream primer is provided with a biotin label.

2. A visual detection method for detecting avian viruses is characterized in that: it adopts visual gene chip to detect fowl virus;

the chip comprises an aldehyde modified matrix membrane and a probe in the avian virus detection primer probe set of claim 1; the substrate film is a silicon wafer or glass;

the detection method comprises the following steps:

A) DNA amplification: amplifying sample DNA by using primers in the avian virus detection primer probe set of claim 1, and mixing products after amplification to form a sample mixture;

B) and (3) hybridization: modifying the sample mixed solution at 100 ℃ for 5 minutes, immediately carrying out ice bath for 3 minutes, dropwise adding the sample mixed solution to a chip array area, standing the chip array area at 35-55 ℃ for 60-150 minutes, washing the chip array area with ultrapure water, and airing the chip array area at room temperature;

E) interpretation: if black spots appear in the corresponding probe area, the detection is positive;

the visual detection method for detecting the avian viruses is used for non-disease diagnosis.

3. The detection method according to claim 2, characterized in that: the standing temperature in step B) was 40 ℃ and the standing time was 120 min.

4. The detection method according to claim 2, characterized in that: the Nanogold-Streptavidin Streptavidin solution concentration of step C) was 4. mu.g/mL.

5. The detection method according to claim 2, characterized in that: the standing time in step D) was 5 min.