CN111020709B - Gene chip and kit - Google Patents
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- CN111020709B CN111020709B CN201911412453.4A CN201911412453A CN111020709B CN 111020709 B CN111020709 B CN 111020709B CN 201911412453 A CN201911412453 A CN 201911412453A CN 111020709 B CN111020709 B CN 111020709B
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
Abstract
The invention provides a gene chip and a kit. The gene chip comprises a substrate and at least one target nucleic acid probe immobilized on the substrate to form a probe array, each target nucleic acid probe being a concatemer of more than two repeats of the target nucleic acid.
Description
Technical Field
The present invention provides a gene chip, in particular, a gene chip comprising a concatemer of two or more repetitive target nucleic acids as target nucleic acid probes.
Background
The penaeus vannamei is an important economic shrimp variety, plays an important role in regulating the economic structure of fishery and increasing the income of fishermen in coastal areas, and has considerable economic and social benefits. However, there are data showing that the influence of related diseases on fishery production values is inferior to natural disasters such as typhoon flood. The five pathogens, especially white spot virus disease (white spot syndrome virus, WSSV), acute hepatopancreatic necrosis disease Acute hepatopancreatic necrosis disease, AHPND), subcutaneous and hematopoietic necrosis virus disease (Infectious hypodermal and hematopoietic necrosis virus, IHHNV), liver and intestine cell worm (Enterocytozoon hepatopenaei, EHP) and blood iridovirus (Shrimp hemocyte iridescent virus, SHIV) are responsible for mass death and yield reduction of penaeus vannamei in many areas. Once the prawns are invaded by the pathogens, the prawns cannot be cured, and the pathogens have strong transmission capability and high transmission speed, and even if extremely trace pathogens exist in the culture environment, the prawns can be rapidly proliferated under proper conditions, so that a large amount of loss is caused. Therefore, it is extremely critical to prevent the shrimp larvae carrying the pathogens from entering the culture environment from the source, and accurate, efficient and sensitive detection means are required to detect the shrimp larvae carrying the pathogens, and timely treatment is required to detect the larvae carrying the pathogens. Therefore, the establishment of the rapid, convenient, economical, efficient and sensitive detection technology of the five pathogens has important significance for guiding the disease prevention and control work.
The seed detection is often required to be quick, efficient, comprehensive and accurate. The currently used nucleic acid detection means are far from meeting these demands. There are also great limitations to various molecular detection means: the national standard method and industry standard for detecting various pathogens are mostly carried out by PCR or nested PCR. However, methods employing PCR tend to be less sensitive; the nested PCR method has complicated steps, single detection index and easy generation of nucleic acid pollution. The fluorescence quantitative PCR method has the same disadvantages: the dye method is used for detecting that the prawn extraction step cannot be controlled in quality, and when the index is more, the error reading is easy to cause by adopting the dissolution curve method; the detection index is small by using a probe rule, and for most four-channel fluorescent quantitative PCR instruments, more than four pathogens can not be detected by a single tube. In addition, the fluorescent probe and the instrument have higher cost, and are not beneficial to large-scale popularization. Therefore, a product is needed in the market, and a plurality of prawn pathogens can be detected rapidly, conveniently and accurately at one time.
Disclosure of Invention
One aspect of the present invention provides a gene chip comprising a substrate and at least one target nucleic acid probe immobilized on the substrate to form a probe array, each target nucleic acid probe being a concatemer of two or more repeats of the target nucleic acid.
In a specific embodiment, the target nucleic acid has 2 repeats and the length of a single target nucleic acid repeat is 12bp to 23bp.
In a specific embodiment, the substrate is selected from one of a nylon membrane, a nitrocellulose membrane, a polyvinylidene fluoride (PVDF) membrane, and a quartz glass substrate.
In a specific embodiment, the gene chip further comprises hybridization positive quality control probes and/or DNA positive quality control probes immobilized on the substrate, wherein the hybridization positive quality control probes are used for detecting the effectiveness of the hybridization process and the DNA positive probes are used for detecting the effectiveness of the process before hybridization.
In a specific embodiment, the target nucleic acid is selected from at least one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, wherein SEQ ID No.1 is used for detecting shrimp white spot disease virus, SEQ ID No.2 is used for detecting shrimp acute hepatopancreatic necrosis disease, SEQ ID No.3 is used for detecting parashrimp subcutaneous and hematopoietic tissue necrosis virus, SEQ ID No.4 is used for detecting shrimp liver and intestine cytozoon, and SEQ ID No.5 is used for detecting shrimp blood iridovirus.
In a specific embodiment, the nucleotide sequence of the hybridization positive quality control probe is shown as SEQ ID No.6, and the nucleotide sequence of the DNA positive quality control probe is shown as SEQ ID No. 7.
The second aspect of the present invention provides a gene chip kit comprising the gene chip according to any one of claims 1 to 7, and a pair of primers for amplifying a fragment containing the target nucleic acid using a DNA of a sample to be tested as a template, wherein the amplified primer for a single strand capable of complementary pairing with the target nucleic acid is labeled with one of biotin, digoxin and horseradish peroxidase.
In a specific embodiment, the pair of primers comprises at least one of SEQ ID No.8/SEQ ID No.9, SEQ ID No.10/SEQ ID No.11, SEQ ID No.12/SEQ ID No.13, SEQ ID No.14/SEQ ID No.15, and SEQ ID No.16/SEQ ID No.17, wherein,
SEQ ID No.8/SEQ ID No.9 is used for prawn white spot disease virus;
SEQ ID No.10/SEQ ID No.11 is used for acute hepatopancreatic necrosis of prawns;
SEQ ID No.12/SEQ ID No.13 is used for treating the submucosal and hematopoietic tissue necrosis viruses;
SEQ ID No.14/SEQ ID No.15 is used for prawn liver and intestine cyst;
SEQ ID No.16/SEQ ID No.17 is used for shrimp blood iridovirus.
In a specific embodiment, the kit further comprises a pair of DNA positive control primers for amplifying a fragment capable of complementary pairing with a DNA positive control probe.
Also included in one embodiment is a hybridization cation control nucleic acid for complementarily pairing with a hybridization cation control probe.
In one embodiment, the pair of DNA cation control primers has the nucleic acid sequences shown in SEQ ID No.18 and SEQ ID No. 19.
In one embodiment, the sequence of the hybrid cation control nucleic acid is shown in SEQ ID No. 20.
In the present invention "/" is indicated as a pair of the former and latter, for example, SEQ ID No.16 and SEQ ID No.17 constitute a pair of primers.
The invention has the beneficial effects that:
the sensitivity of the gene chip using the reverse dot hybridization method has not been high, and although increasing the probe length can effectively increase the sensitivity of the gene chip, nonspecific signals are also particularly easy to generate, thus limiting the development of such gene chip technology. The invention prepares the detection probe by utilizing a mode of connecting two or more nucleotide sequences in series, can effectively improve the detection sensitivity, and can avoid the phenomenon of poor specificity caused by overlong probe length due to proper series times.
The serial probe is applied to detecting the pathogen of the penaeus vannamei, realizes that five pathogens to be detected objects are simultaneously amplified by PCR for detecting a gene chip, has very high sensitivity and specificity, is convenient, simple and quick to use, can complete all detection procedures including extraction and amplification in three hours, and has quite high practical value.
Drawings
Fig. 1 shows a schematic layout of probes on a chip.
FIG. 2 shows the results of five pathogens, AHPND, WSSV, EHP, IHHNV and SHIV, detected using one of the probes of the invention.
Fig. 3 shows another schematic layout of probes on a chip.
Fig. 4 shows the results of detection of five pathogens AHPND, WSSV, EHP, IHHNV and SHIV using different AHPND probes.
Detailed Description
The invention is further illustrated below with reference to the examples, which are merely illustrative of the invention and do not constitute a limitation of the invention in any way.
The reagents used in the examples below are all commercially available unless otherwise specified.
Example 1
1. Primer, probe design
Primers and probes are designed according to the specific gene sequences of five pathogens in NCBI gene database, referring to Primer-BLAST primers and probe design principle. The primer length is about 20-23nt. Because the PCR reaction of the invention has a large number of weights, a large number of primers and probes are designed and synthesized in the early working process of the invention, and particularly, the primers and probes with high sensitivity and good specificity are screened from the primers and probes by using the sequence repeat probes.
PC: the sequence of the positive primer of the shrimp genome 18s rDNA is shown as SEQ ID No.18, and biotin is modified at the 5' end; the reverse primer sequence is shown as SEQ ID No. 19; the DNA positive quality control Probe (PC) sequence is shown in SEQ ID No. 7.
HC: the hybridization positive quality control nucleic acid sequence is shown as SEQ ID NO.20, and biotin is modified at the 5' end; the sequence of the hybridization positive quality control probe (HC) is shown in SEQ ID No. 6.
The forward primer sequence of the AHPND pirB gene is shown as SEQ ID No.10, and biotin is modified at the 5' end; the reverse primer sequence is shown as SEQ ID No. 11; the single repeat probe sequence is shown in SEQ ID No. 2.
The EHP 18s rDNA forward primer sequence is shown as SEQ ID No.14, and biotin is modified at the 5' end; the reverse primer sequence is shown as SEQ ID No. 15. The single repeat probe sequence is shown in SEQ ID No. 4.
The WSSV vp28 gene forward primer sequence is shown as SEQ ID No.8, and biotin is modified at the 5' end; the reverse primer sequence is shown as SEQ ID No. 9; the single repeat probe sequence is shown in SEQ ID No. 1.
The IHHNV specific CDS (Sequence coding for amino acid sin protein) gene forward primer sequence is shown as SEQ ID No.12, and biotin is modified at the 5' end; the reverse primer sequence is shown as SEQ ID No. 13; the single repeat probe sequence is shown in SEQ ID No. 3.
The forward primer sequence of the SHIV specific amplified target specific CDS (Sequence coding for amino acid sin protein) gene is shown as SEQ ID No.16, and biotin is modified at the 5' end; the reverse primer sequence is shown as SEQ ID No. 17; the single repeat probe sequence is shown in SEQ ID No. 5.
Primers, single repeat probe of probes, two repeat probes and three repeat probes were synthesized by the biological company limited and probes were synthesized by the english-strapdesk biological company limited.
Amplification of PCR products
PCR amplification reagents (10 Xbuffer, taq enzyme, UNG, dUTP and dNTP) were purchased from Dalianbao Biotechnology Co., ltd.
The reaction system was prepared in the proportions shown in Table 1.
TABLE 1
And (3) after shaking and uniformly mixing the prepared PCR reaction system, adding 3 mu L of DNA template for PCR amplification. Wherein, the amount of each upstream primer is about 2 times that of the downstream primer, so as to amplify a large amount of single strands complementarily paired with the probe, thereby omitting the subsequent denaturation step.
The amplification procedure was:
50℃2min
94℃4min；
(94 ℃ C. 30s,53 ℃ C. 30s,72 ℃ C. 10 s), 40 cycles;
(94 ℃ C. 30s,68 ℃ C. 30s,72 ℃ C. 10 s), 3 cycles;
preserving at 4 ℃.
The amplified PCR product was used for the next chip hybridization without purification.
3. Preparation of prawn pathogen gene chip
Nitrocellulose membranes were purchased from Sidoris, germany.
1) Cutting a positively charged nylon film into film strips;
2) Soaking the cut membrane strip in sterilized distilled water for 10min, and then suspending and airing;
3) Each probe was dissolved in a probe buffer (composition: 0.4N NaOH,0.1M EDTA) to prepare a 4. Mu.M solution to obtain a probe solution, and sequentially immobilizing 2. Mu.L of the probe solution on a nylon membrane;
4) And irradiating the wet film with the probe for 5min under ultraviolet, and taking out and naturally drying.
PCR reverse dot hybridization
The nucleic acid hybridization solution I, the nucleic acid hybridization solution II, the hybridization washing solution, the color development solution and the nucleic acid hybridization lattice are all from Jiangsu Hunting biological technology Co. Microplate thermostats were purchased from auspicious biology ltd.
1) Placing the nucleic acid hybridization solution I and the nucleic acid hybridization solution II in a water bath kettle to be preheated to 45 ℃ for standby, wherein the component of the nucleic acid hybridization solution I is 6 XSSC, 2.5% skimmed milk powder and 1% SDS, and the component of the hybridization solution II is alkaline phosphatase marked streptavidin solution;
2) Setting the temperature of the microplate constant-temperature oscillator to 45 ℃ in advance, so that the temperature in the microplate constant-temperature oscillator reaches the optimal hybridization temperature of 45 ℃;
3) Taking a prawn pathogen gene detection chip membrane by using sterile forceps, putting the membrane into a centrifuge tube, and adding 600 mu L of preheated nucleic acid hybridization solution I for standby;
4) Adding the PCR product obtained in the section 4 into the crystal lattice with the chip obtained in the section 5, and hybridizing for 10min in a constant temperature oscillator at 1500rpm and 45 ℃;
5) After hybridization, 1. Mu.L of preheated nucleic acid hybridization solution II is added, and the mixture is oscillated for 3min in a constant temperature oscillator at 1500rpm and 45 ℃;
6) Discarding hybridization solution in lattice, adding 2ml preheated hybridization washing solution with hybridization washing solution component of 2 XSSC, washing in constant temperature shaker at 1500rpm and 45 deg.C for 2min, repeating twice;
7) Discarding the washing solution in the crystal lattice, adding 500 mu L of pre-heated color developing solution, wherein the color developing solution comprises premixed solution of BCIP and NBT, and developing color for 3min in a constant-temperature oscillator at 1500rpm and 45 ℃;
8) Interpretation of the results: and directly judging in a crystal lattice, hybridizing a quality control probe position, and indicating that brown bright spots appear on the whole quality control probe position to be effective in detection, wherein the brown bright spots appear on the corresponding probe position, namely the corresponding pathogen is contained.
Example 2
Acquisition of the genome of prawns
Extracting genome of the prawn by using an animal tissue extraction kit from Jiangsu Hunting biological technology Co., ltd, screening out prawns which do not carry AHPND, WSSV, EHP, IHHNV and SHIV five pathogens from a prawn sample by using a method in the prior art, and finally obtaining a prawn genome solution which does not carry AHPND, WSSV, EHP, IHHNV and SHIV five pathogens.
Among them, the method for detecting AHPND pathogens is OIE recommended method (Sirintip Dangtipa, ratchanok Sirikharinb, piyachat Sanguanrut, et al, AP4method for two-tube nested PCR detection of AHPND isolates of Vibrio parahaemolyticus [ J ]. Aquaculture Reports,2015, 2:158-162.); methods for detecting WSSV are part 2 of the GB/T28630.2-2012 white spot syndrome (WSD) diagnostic protocol: nested PCR detection method [ S ]; the method for detecting IHHNV is a GB/T25878-2010 method for detecting infectious subcutaneous and hematopoietic necrosis virus (IHHNV) PCR [ S ]; the method of detecting EHP is the ethernet water center Network (NACA) recommended method (NACA (2018). A New and Improved PCR detection method forEnterocytozoonhepatopenaei (EHP) based on a gene encoding a spore wall protein, published by the Network of Aquaculture Centres in Asia-Pacific, bangkok, thailand); the method of detecting SHIV is likewise the method recommended by the national center for Care for Ethernet (NACA) (Qia, liang, chen, meng-Meng, wan, xiao-Yuan, et al Charabacteria of a new member of Iridoviridae, shrimp Hemocyte Iridescent Virus (SHIV), found in white leg shrimp (Litopenaeus vannamei) [ J ]. Scientific Reports,7 (1): 11834).
Example 3
Detection of test plasmid Standard
Amplifying the pirB gene of AHPND, vp28 gene of WSSV, 18s rDNA gene of EHP, specific viral CDS region of IHHNV and specific gene fragment of specific viral CDS region of SHIV respectively, inserting the fragments of all pathogens into pMD19-T vector respectively, transferring the connection product into DH5 alpha competent cells, culturing, detecting the inserted target fragment by PCR and sequencing (Shanghai) after new bacterial colony is grown, screening to positive bacterial colony, and extracting positive plasmid to obtain plasmid standard. Wherein, the plasmid standard of the AHPND is called an AHPND plasmid standard; the plasmid standard for WSSV is called a WSSV plasmid standard; the plasmid standard for EHP is referred to as EHP plasmid standard; the plasmid standard for IHHNV is referred to as IHHNV plasmid standard; the plasmid standard for SHIV is referred to as SHIV plasmid standard.
Determining the concentration and purity of positive plasmid DNA by using an ultra-micro spectrophotometer, calculating the copy number concentration of plasmid DNA contained in the unit volume of the extracted sample according to Moore's law, and finally diluting the plasmid standard of each pathogen to 3 fg. Mu.L by using the prawn genome solution (without carrying AHPND, WSSV, EHP, IHHNV and SHIV five pathogens) prepared in example 2 -1 To be used as DNA templates for PCR amplification of each pathogen, respectively; in addition, the solution of the plasmid standard substance of each pathogen prepared by using the prawn genome solution (without carrying AHPND, WSSV, EHP, IHHNV and SHIV pathogens) prepared in example 2 was used as a DNA template for PC PCR amplification, wherein the concentration of the plasmid standard substance of each pathogen was 3 fg. Mu.L -1 . Wherein, plasmid copy number= [ plasmid concentration (ng. Mu.L) -1 ) Xplasmid solution volume (. Mu.L). Times.6.02X 10) 23 ]/(plasmid length bp+insert (i.e., target nucleic acid) length bp). Times.660 g.mol -1 ]。
mu.L of the PCR DNA template prepared as above was added to the reaction system of Table 1 of section 2 of example 1, respectively, followed by PCR amplification according to the procedure of section 2 of example 1, to obtain PCR amplified products of five pathogens, respectively. Meanwhile, 3. Mu.L of the prawn genome solution was added to the reaction system of the section 1 of example 1, and then PCR amplification was performed according to the procedure of the section 2 of example 1, to obtain a PC PCR amplification product.
Based on the detection of five pathogens of prawns, the gene chip of the pathogens of the prawns is designed into a 4 multiplied by 3 array. In the array, probes were immobilized on nitrocellulose membranes according to the procedure of section 3 of example 1 in the arrangement shown in FIG. 1, with each pathogen probe being two duplicate probes. The method comprises PC and HC 2 positive quality control points and 5 pathogen detection points, wherein the total number of the PC and HC 2 positive quality control points is 7, and the PC is DNA positive quality control points and is used for detecting 18s rDNA genes in shrimp genome so as to monitor the effectiveness of the extraction and PCR amplification process of sample DNA; HC is the positive control point of hybridization and the hybridization process is monitored.
The HC hybrid positive control nucleic acid, PC PCR amplification products, and PCR amplification products of the five pathogens above were detected in triplicate using the gene chip arrangement as in fig. 1. The results are shown in FIG. 2.
The results of fig. 2 show that the detection signal points are all clearly indicative of the corresponding pathogen, without non-specific signal points.
Example 4
Detection effect of probes of different repetition degrees
Gene chips containing 1 repeat, 2 repeat, 3 repeat, probes of equal length to the probe of AHPND 2 repeat, respectively (synthesized by Bio Inc. as shown in SEQ ID.21) were prepared to determine the sensitivity and specificity of the probes at different degrees of repeat. The layout of the four AHPND gene chips is shown in FIG. 3, wherein HC is used as a hybridization positive quality control point to monitor the hybridization process. The detection subjects were PCR amplification products of the five pathogens of example 3, respectively. The results are shown in FIG. 4.
The results in FIG. 4 show that at a template concentration of 3 fg/. Mu.L AHPND plasmid standard, a single duplicate probe failed to detect signal, indicating its lower sensitivity. Whereas 2 and 3 replicates of probes all showed significant positive signals; however, among the 3 duplicate probes, detection using PCR amplification products of the other four pathogens showed false positive signals; in addition, there are false positive signals of other four pathogen PCR amplification products as well as two duplicate equal length probes.
It is preferable to use a tandem sequence of 2 repeats as a probe.
Example 5
Detection of test samples
The tissue sample of the prawn carrying AHPND, WSSV, EHP and SHIV four pathogens is derived from yellow sea institute of China aquatic sciences, and is a blind test sample for verifying the pathogen detection capability of the prawn in 2019 of national agricultural rural area. A total of 20 samples. 8 prawn tissue samples are obtained by local sampling in Jiangsu like east.
The animal tissue extraction kit is from Jiangsu shotgun biotechnology limited company and is used for extracting genomes in the 28 samples to be detected respectively.
The extracted genome is used as a DNA template, added into the reaction system in the table 1 of the section 2 of the example 1 respectively, and then PCR amplification is carried out according to the procedure in the section 2 of the example 1 to obtain PCR amplification products of the sample to be detected, and the detection is carried out by using the gene chip prepared in the example 3.
In addition, five pathogens were also tested using the prior art technique of example 2.
Wherein, 6 prawn samples to be tested are used for detecting whether AHPND pathogens exist, 4 prawn samples to be tested are used for detecting whether WSSV pathogens exist, 8 prawn samples to be tested are used for detecting whether IHHNV pathogens exist, and 6 prawn samples to be tested are used for detecting whether EHP pathogens exist.
The results are shown in Table 2. As can be seen from Table 2, the results of the detection of the gene chip of the 2 repetitive probes of the present invention completely match the results of the detection of the prior art.
TABLE 2
| Detection index | Total number of samples | Nest type PCR positive sample number | Gene chip method positive sample number |
| AHPND | 6 | 4 | 4 |
| WSSV | 4 | 2 | 2 |
| IHHNV | 8 | 8 | 8 |
| EHP | 6 | 4 | 4 |
| SHIV | 4 | 2 | 2 |
Although the invention has been described with reference to specific embodiments, those skilled in the art will appreciate that various modifications might be made without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of materials, and method to the essential scope, spirit, and scope of the present invention. All such modifications are intended to be included within the scope of this invention as defined in the following claims.
Sequence listing
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Claims (5)
1. A gene chip comprising a substrate and at least one target nucleic acid probe immobilized on the substrate to form a probe array;
the target nucleic acid has 2 repeats, and the length of a single target nucleic acid repeat is 12bp to 23bp;
the target nucleic acid is selected from at least one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, wherein the SEQ ID No.1 is used for detecting white spot disease virus of the prawns, the SEQ ID No.2 is used for detecting acute hepatopancreatic necrosis disease of the prawns, the SEQ ID No.3 is used for detecting necrosis virus of tissues under the prawns and hematopoietic tissues, the SEQ ID No.4 is used for detecting liver and intestine cell worms of the prawns, and the SEQ ID No.5 is used for detecting iridovirus of blood of the prawns;
the gene chip further comprises a hybridization positive quality control probe and/or a DNA positive quality control probe which are/is fixed on the matrix, wherein the hybridization positive quality control probe is used for detecting the effectiveness of a hybridization process, and the DNA positive probe is used for detecting the effectiveness of a process before hybridization;
the nucleotide sequence of the hybridization positive quality control probe is shown as SEQ ID No.6, and the nucleotide sequence of the DNA positive quality control probe is shown as SEQ ID No. 7.
2. The gene chip of claim 1, wherein the substrate is one selected from the group consisting of nylon membrane, nitrocellulose membrane, polyvinylidene fluoride membrane, and quartz glass substrate.
3. A gene chip kit comprising the gene chip according to claim 1 or 2, and a pair of primers for amplifying a fragment containing the target nucleic acid using a DNA of a sample to be tested as a template, the amplified primer for a single strand capable of complementary pairing with the target nucleic acid being labeled with one of biotin, digoxin and horseradish peroxidase;
the pair of primers comprises at least one pair of SEQ ID No.8/SEQ ID No.9, SEQ ID No.10/SEQ ID No.11, SEQ ID No.12/SEQ ID No.13, SEQ ID No.14/SEQ ID No.15, and SEQ ID No.16/SEQ ID No.17, wherein,
SEQ ID No.8/SEQ ID No.9 is used for prawn white spot disease virus;
SEQ ID No.10/SEQ ID No.11 is used for acute hepatopancreatic necrosis of prawn;
SEQ ID No.12/SEQ ID No.13 is used for treating the submucosal and hematopoietic tissue necrosis viruses;
SEQ ID No.14/SEQ ID No.15 is used for prawn liver and intestine cyst;
SEQ ID No.16/SEQ ID No.17 is used for shrimp blood iridovirus.
4. The kit according to claim 3, further comprising a pair of DNA positive quality control primers for amplifying a fragment complementarily paired with the DNA positive quality control probe; and/or
The kit also comprises hybridization cation quality control nucleic acid for complementary pairing with the hybridization cation quality control probe.
5. The kit according to claim 4, wherein the pair of DNA cation control primers has the nucleic acid sequences shown in SEQ ID No.18 and SEQ ID No. 19; and/or
The sequence of the hybridization cation control nucleic acid is shown as SEQ ID No. 20.
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| CN114182031A (en) * | 2021-12-22 | 2022-03-15 | 江苏猎阵生物科技有限公司 | Nucleic acid probe and application thereof |
| CN114214393A (en) * | 2021-12-30 | 2022-03-22 | 上海百傲科技股份有限公司 | Universal nucleic acid microarray chip and detection method |
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