CN111534639A

CN111534639A - African swine fever gene chip and application thereof

Info

Publication number: CN111534639A
Application number: CN202010373798.XA
Authority: CN
Inventors: 孙坚; 王夕冉; 王衡; 张桂红; 刘雅红; 廖晓萍
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2020-05-06
Filing date: 2020-05-06
Publication date: 2020-08-14

Abstract

The invention provides a sequence combination of African swine fever virus, which comprises a DNA probe set, wherein the DNA probe set comprises SEQ ID NO.7, SEQ ID NO.14, SEQ ID NO.24, SEQ ID NO.31, SEQ ID NO.40, SEQ ID NO.73, SEQ ID NO.74, SEQ ID NO.77, SEQ ID NO.78, SEQ ID NO.84, SEQ ID NO.96, SEQ ID NO.130, SEQ ID NO.134, SEQ ID NO.142 and SEQ ID NO. 153. Preferably, the DNA probes comprise 155 DNA probes, and the sequences of the DNA probes are shown in SEQ ID NO.1-SEQ ID NO. 155. With the preferred embodiment of the present invention, a detection rate of 100% of the mutation sites can be achieved.

Description

African swine fever gene chip and application thereof

The technical field is as follows:

the invention belongs to the field of virus target capture sequencing, particularly relates to capture of an African swine fever key sequence, and particularly relates to design and application of a liquid-phase probe for capturing the African swine fever virus key sequence target by a trocar.

Technical background:

african Swine Fever (ASF) is an acute, virulent swine infectious disease infected by African swine fever virus, with bleeding and fever as the main symptoms. The African swine fever has short disease process and extremely high mortality rate. African swine fever is caused by African Swine Fever Virus (ASFV), a double-stranded DNA virus that is the only member of the genus African virus of the family African viridae, whose primary infection is in domestic or wild pigs. The African swine fever virus whole genome is about 170-193kb in length, and 150-167 open reading frames are detected in different strains.

With the development and maturation of high-throughput sequencing technology, whole genome scanning of virus samples based on whole genome sequencing has become a powerful means for studying genetic evolution rules of viruses. Because the acquisition of single African swine fever virus DNA is difficult, the current general sequencing strategy is to extract the total DNA of host and virus, perform second-generation sequencing and library building through enough DNA, extract a very small amount (about 0.75%) of virus short-reading sequences from the final mass data, and perform assembly and splicing to obtain the whole genome sequence of the virus.

The second-generation sequencing based on the liquid-phase probe has extremely high specificity, can be used for capturing and sequencing the whole virus genome in the nucleic acid of the tissue sample, effectively reduces the sequencing cost, improves the sequencing depth and more accurately discovers the genetic variation information of the African swine fever virus.

Therefore, the method which is low in price and high in accuracy is developed to quickly obtain the accurate variation of the virus genome, and the method has profound significance for researching the global evolution network of the African swine fever virus.

The invention content is as follows:

the inventor provides a group of gene probes by analyzing the whole genome data of the existing classical swine fever virus, and realizes the capture sequencing of the gene sequence of the African classical swine fever virus through a gene chip comprising the gene probes.

Thus, in a first aspect, the present invention provides a combination of sequences of african swine fever virus comprising a set of DNA probes comprising SEQ ID No.7, SEQ ID No.14, SEQ ID No.24, SEQ ID No.31, SEQ ID No.40, SEQ ID No.73, SEQ ID No.74, SEQ ID No.77, SEQ ID No.78, SEQ ID No.84, SEQ ID No.96, SEQ ID No.130, SEQ ID No.134, SEQ ID No.142, SEQ ID No. 153.

In one embodiment, the DNA probe set comprises 155 DNA probes, and the sequence of the DNA probes is shown in SEQ ID NO.1-SEQ ID NO. 155.

In one embodiment, the DNA probe further comprises a linker sequence.

In one embodiment, the DNA probe is a biotin-labeled DNA probe.

In a second aspect, the invention provides an African swine fever virus detection kit, comprising a set of DNA probes comprising SEQ ID No.7, SEQ ID No.14, SEQ ID No.24, SEQ ID No.31, SEQ ID No.40, SEQ ID No.73, SEQ ID No.74, SEQ ID No.77, SEQ ID No.78, SEQ ID No.84, SEQ ID No.96, SEQ ID No.130, SEQ ID No.134, SEQ ID No.142, SEQ ID No. 153.

In one embodiment, the DNA probe is a biotin-labeled DNA probe.

In a third aspect, the present invention provides a method for screening african swine fever virus gene mutation by using a gene chip, the method comprising:

1) extracting the genome DNA of a sample to be detected;

2) the DNA is broken to the range of 200-300 bp;

3) preparing a DNA fragment library from the fragmented genomic DNA;

4) the DNA fragment library is hybridized with the sequence combination of the first aspect of the present invention and the gene chip of the second aspect to detect gene mutation.

In a fourth aspect, the invention also provides the use of the sequence combination of the first aspect and the gene chip of the second aspect for detecting African swine fever.

The method and the gene chip have the following advantages:

compared with whole genome sequencing, the method and the chip greatly save the required sequencing data amount; the method and the chip of the invention can detect most of mutations of disease-related pathogenic genes at one time, balance the cost performance and have great significance for the rapid diagnosis, prevention and control of African swine fever. The chip of the invention carries out gene capture sequencing of African swine fever virus by a liquid phase probe technology, and is convenient, rapid and accurate. The second-generation sequencing based on the liquid-phase probe has extremely high specificity, can be used for capturing and sequencing the whole virus genome in the nucleic acid of the tissue sample, effectively reduces the sequencing cost, improves the sequencing depth and more accurately discovers the genetic variation information of the African swine fever virus.

The specific implementation mode is as follows:

the objects and functions of the present invention and methods for accomplishing the same will be apparent by reference to the exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below, and may be implemented in various forms. The nature of the description is merely to assist those skilled in the relevant art in a comprehensive understanding of the specific details of the invention.

What has been regulated by interferon in the genome of ASFV is a multigene family gene (MGF), and the low-toxicity ASFV lacks different copy numbers of these genes in MGF360 or MGF505 families. Deletion of these genes in the ASFV genome results in increased production of type I interferons, suggesting their important role in the regulation of their levels. A small but significant genetic diversity was found in the MGF505-2R gene, indicating that ASFV molecules evolve slowly, which plays an important role in regulating the production of interferon I and the phenomenon of blood adsorption. In addition, the protein encoded by I329L is used as an inhibitor of toll-like receptor 3 signal pathway and is involved in I-type interferon induction and response inhibition. In ASFV-infected feral pig lung (WSL) cells, the product of the K145R gene proved to be the most significant viral protein. Recent transcriptome analysis of infected pigs showed that K145R was abundantly expressed at the RNA level. pK145R was also identified as an antibody-inducing protein in convalescent pigs by screening of the viral cDNA expression library.

The ASFV capsid is composed of 17280 proteins, including 1 major protein (p72) and 4 minor proteins (pM1249L, p17, p49 and pH 240R). And pM1249L protein with the length of 100 nanometers is taken as a core organizer to form an intermolecular network with other capsid proteins to form a capsid framework. KP177R (P22) is an early induction structural protein that is detectable on the cell membrane by immunolabeling early after viral infection. CP204L encodes phosphoprotein, interacts with host cells, binds ribonucleoprotein-K, and is involved in virus entry. NP419L encodes a DNA ligase that is an integral part of the mechanism of the minimal DNA substrate excision repair pathway (BER) of ASFV and acts on damaged DNA in a short time. D1133L is one of the 6 helicase superfamily members encoded by ASFV and has been reported to play a role in transcription. QP383R encodes an enzyme involved in redox metabolism, being a bacterial NifS protein homologue.

Therefore, the method is particularly important for detecting important gene mutation in ASFV genome. The invention solves the problem of how to realize more effective detection by using the simplest probe sequence.

In the present invention, the reference genome of an african swine fever virus is MK333180.

The gene chip comprises probes aiming at the following genes: MGF _360-1L, KP177R, MGF _110-1L, MGF _505-2R, MGF _505-9R, K145R, M1249L, C84L, CP204L, NP419L, D1133L, QP383R, I267L, I329L & I73R and MGF _ 360-16R.

The gene probe sequence is as follows:

ASF was originally reported in kenya in 1921. Introduced into the grurgia republic in 2007 and later propagated to other eastern european countries, including russia (2007), ukraine (2012), russia (2013), ridorania (2014), estonia (2014), poland (2014), latanova (2014), ralvaia (2014), romania (2017), the czech republic (2017), hungarian (2018), belgium (2018) and china (2018).

Since 9 months in 2018 when ASF introduced Belgium, the entire genome sequence of African swine fever virus in east Europe is almost identical to that obtained in China. The reported ASFV key gene sequence is compared with the reference genome sequence to obtain the mutation site.

In 2007, one ASF case was reported in Gruggium, with the virus genome being GA/2007(GenBank: FR682468.1), and since then, ASFV became epidemic in Russia and spread to other European countries.

2013 to 2018, in russia ASF. Sampling studies were conducted in several regions, and the biological properties of isolate Odintsov 02/14 (GenBank: KP843857) were significantly different from those of other strains, and virus antibodies were detected in 71.4% of animals, with a mortality rate of 87.5%. The Odintsovo02/14 isolate was from a wild boar and was sampled in the Tarakanovsky forest region of Odintsovo region in Moscow.

In 2018, 8 and 3 days, the first swine fever case was found in a certain pig farm in suburbs of Shenyang, Liaoning province. The viral genome of ASFV, which is responsible for the first ASF outbreak in China, was detected in swine with the first ASF outbreak in Shenyang, and the whole genome sequence of ASFV-SY18 (GenBank: MH766894.1) was uploaded by Miao et al. It was also reported that the sequence had the highest similarity to the ASFV whole genome sequence isolated in Poland in 2017, which is called PoL/2017(GenBank: MG 939588.1).

And (3) comparing the reported ASFV key gene sequence with the reference genome sequence to obtain a mutation site.

The mutation sites of the genes selected in this application on the reference genome are as follows:

in the present invention, mutations are expressed using methods commonly used in the art. "g." denotes the genomic sequence. Mutations are indicated by the symbol ">", such as: 123a > T, indicating that a at position 123 is replaced with T compared to the reference sequence; deletions are denoted by "del"; such as: 2052delA, which indicates the deletion of A at position 2052 compared to the reference sequence; insert is denoted as "ins"; such as: g.5756-5757 insAGG, which indicates that three bases of AGG are inserted between positions 5756 and 5757 as compared to the reference sequence.

For purposes of the present specification and claims, reference to gene sequences will be understood by those skilled in the art to include virtually either or both of the complementary double strands. For convenience, in the present specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. For example, reference to a probe sequence actually includes the sequence and its complement. For example, reference to MK333180.1:876-976 actually includes the complementary sequence thereof. One skilled in the art will also appreciate that one strand may be used to detect the other strand and vice versa.

In this example, probe sequences of 120bp in length are designed from the first base in the 5 'to 3' direction of the coding sequence of the gene according to the principle of reverse complementary sequence, and there is an overlap between every two adjacent probe sequences, which is 1/2 or 2/3 of the length of the probe. For the length of the probe sequence, more than 120bp brings difficulty in synthesis, less than 110bp reduces the capture capacity and increases the number of probes, so that the cost is increased, the balance of the two is very important, and the inventor obtains a better value of 120bp through continuous tests. For the overlap of the probe between each two adjacent probe sequences, the overlap between each two adjacent probe sequences is 1/2 or 2/3 of the length of the probe, because 2 or 3 layers of probe coverage will be formed for each region, so the probe coverage is uniform, and thus the uniformity of capture is not affected, and the uniformity of capture is affected by the non-uniform probe coverage. Thus, the overlap between each two adjacent probe sequences is 1/2 or 2/3 of the length of the probe. In the case where the overlap between each two adjacent probe sequences is 1/2 the length of the probe, preferably the length of the probe is even; in the case where the overlap between each two adjacent probe sequences is 2/3 times the length of the probe, it is preferred that the probe length is an integral multiple of 3. However, where the probe length is not an even number or an integer multiple of 3, it is also possible that the overlap is taken to be an approximate integer of the probe length 1/2 or 2/3, although the effect is not as good as if the probe length is an even number or an integer multiple of 3. Gene chips were prepared by commercial Gene chip corporation and each probe sequence was replicated three times.

Example (b):

1. experimental methods

The Affymetrix HG U133A chip platform is used for detecting African swine fever samples to test the gene chip (comprising the probes shown in the sequences SEQ ID NO.1-SEQ ID NO.155 and a conventional control sequence). Gene chip design and testing was performed by Ejiekang (Beijing) Biotechnology, Inc.

The first step is as follows: extraction of genome

The whole Blood genome of the jugular vein, inferior vena cava, auricular vein or heart of an African swine fever sample was extracted using the DNeasy Blood & Tissue Kit (QIAGEN, Germany) according to the instructions provided by the manufacturer.

The second step is that: quality control and quantification of genome

The sample genome was subjected to concentration measurement using Qubit, and only double-stranded DNA was measured, and the amount of double-stranded DNA was calculated from the concentration and volume. When the DNA purity is analyzed by a spectrophotometer, the A260/A280 should be close to 1.8 and be relatively pure DNA (i.e., the ratio is 1.7-1.9). The samples were stored at-20 ℃.

The third step: random disruption of DNA

Covaris was used to randomly disrupt the sample genome to 150-200 bp.

The fourth step: genomic library construction

And (3) performing end repair on the interrupted genome DNA, adding ' A ' to the 3 ' end, then connecting a joint, and finally performing sample marking and DNA enrichment by using a PCR amplification method. The linker sequences include SEQ ID No.156 and SEQ ID No. 157:

SEQ ID NO.156:ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNT

SEQ ID NO.157:NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC。

the fifth step: biotin labels

The DNA was labeled with Random Primer DNA Labeling Kit type B (Biotin Labeling).

1. The following reagents were added to a 1.5mL RNase-free centrifuge tube according to the following table:

reagent	Dosage of
		Template DNA	50-150ng
2 × random primer labeling reaction solution (B type)	10μL
		Ultrapure water	Add to 15μL

2. Boiling water bath for 5-10 min, or heating at 100 deg.C for 5-10 min in PCR instrument, and immediately freezing.

3. The high speed centrifugation speed of seconds allowed all the liquid to collect at the bottom of the tube, and the following reagents were added:

reagent	Dosage of
		Klenow exo polymerase	1μL
Ultrapure water	4μL

Note: the reaction solution (type B) was included in the 2 Xrandom primer labeling reaction without adding dNTPs.

4. Gently blow, beat and mix evenly. If liquid drops are attached to the tube wall, all liquid is concentrated at the bottom of the tube by high-speed centrifugal speed for second.

5. Keeping the temperature at 37 ℃ for 1-20 hours. The labeling efficiency is related to the amount of template and incubation time, as shown in the following table:

6. the reaction was stopped by adding 1. mu.L of self-contained 0.5M EDTA (pH 8.0).

And a sixth step: hybridization of

20 × eukaryotic hybridization control reagents were heated at 65 deg.C for 5 minutes prior to use. The labeled DNA was heated at 100 ℃ for 5 minutes. Reagents were added to a new 1.5mL RNase-free centrifuge tube according to the following table:

adding a proper volume of 1 Xhybridization buffer solution into the sample adding hole to wet the chip; prehybridization of the chip at 45 ℃ for 10 minutes at 60 rpm; the treated sample was heated at 45 ℃ for 5 minutes and centrifuged at maximum speed for 5 minutes; the buffer was removed from the chip and an equal volume of the treated hybridization solution was added at 45 ℃ to hybridize the chip for 16 hours at 60 rpm.

Chip and method for manufacturing the same	Hybridization volume	Total volume
			Standard type	200μL	250μL
Medium size	130μL	160μL
			Small-sized	80μL	100μL
Miniature size	80μL	100μL

The seventh step: elution and staining

After 16 hours of hybridization, the hybridization solution was removed from the chip and filled into a new 1.5mL centrifuge tube, and stored on ice or at-80 ℃ for a long period of time. The chip was filled with elution buffer a. The following solutions were prepared:

SAPE liquid (prepared before use, stored at 4 ℃):

antibody solution:

composition (I)	Volume of	Final concentration
			2 × MES stain buffer	300.0μl	1×
50mg/ml acetylated BSA	24.0 μl	2mg/ml
			Goat immunoglobulin G standard substance of 10mg/ml	6.0μl	0.1mg/ml
0.5mg/ml biotinylated antibody	3.6μl	3μg/ml
			Deionized water	266.4μl
Total volume	600μl

The elution station operates according to the following table:

eighth step: interpretation of results

The chip was scanned using a GeneChip Scanner 30007G and the image analysis processing of the chip was performed.

2. The experimental results are as follows:

in order to verify the effect of the gene chip of the invention, 180 collected samples were selected by the inventors to detect the mutation of the African swine fever virus. For each sample mutation known, we re-identified the mutation sites by sequencing through the whole genome for inconsistent results of the detection.

Selecting a sample:

each sample is repeated three times, and for all 360 sample detections, the detection rate of gene mutation is determined by using a DNA probe SEQ ID NO.1-SEQ ID NO. 155: the detection efficiency is 100%. In order to simplify the gene chip of the invention, the probes included in the gene chip are optimized by comprehensively considering the cost and the detection rate of the probes, and the detection rate of the mutation sites is 96.8% by using DNA probes SEQ ID NO.7, SEQ ID NO.14, SEQ ID NO.24, SEQ ID NO.31, SEQ ID NO.40, SEQ ID NO.73, SEQ ID NO.74, SEQ ID NO.77, SEQ ID NO.78, SEQ ID NO.84, SEQ ID NO.96, SEQ ID NO.130, SEQ ID NO.134, SEQ ID NO.142 and SEQ ID NO. 153. Without being limited by the theory, the inventors believe that the interference of the host genome sequence can be better eliminated by adding other probe sequences for correction, and a better detection rate is achieved.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

The method is simple and easy to operate, greatly saves the detection time, achieves the aim of rapid detection, reduces the detection cost, and can be used for controlling the spreading of the African swine fever virus, the quarantine of entry and exit animals and byproducts thereof and the large-scale investigation of the African swine fever virus in a pig farm. 1. Successfully constructs an African swine fever detection gene chip: the African swine fever virus probe prepared by the invention can be specifically combined with a target gene of a corresponding virus, and a hybridization signal is strong and stable. 2. The prepared gene detection chip has good methodological characteristics: the invention establishes a gene chip detection method with good specificity, high sensitivity, strong stability and time and labor saving. 3. The construction of the detection gene chip provides a rapid and efficient means for the differential diagnosis of the African swine fever, and provides technical support for the quarantine of the entry and exit animals and the control of corresponding epidemic diseases in the future. The method has very important significance for meeting the requirements of quarantine work of entry and exit animals, controlling the spread of African swine fever viruses and ensuring the safe and healthy development of animal husbandry in China.

Claims

1. A sequence combination for detecting African swine fever virus, the sequence combination comprises a DNA probe, the DNA probe comprises SEQ ID NO.7, SEQ ID NO.14, SEQ ID NO.24, SEQ ID NO.31, SEQ ID NO.40, SEQ ID NO.73, SEQ ID NO.74, SEQ ID NO.77, SEQ ID NO.78, SEQ ID NO.84, SEQ ID NO.96, SEQ ID NO.130, SEQ ID NO.134, SEQ ID NO.142, SEQ ID NO. 153.

2. The sequence combination according to claim 1, wherein the DNA probe set comprises 155 DNA probes, and the sequences of the DNA probes are shown in SEQ ID NO.1-SEQ ID NO. 155.

3. The sequence combination according to claim 1 or 2, wherein the DNA probe further comprises a linker sequence.

4. The sequence combination according to claim 1 or 2, wherein the DNA probe is a biotin-labeled DNA probe.

5. A gene chip for detecting African swine fever virus comprises a DNA probe, wherein the DNA probe comprises SEQ ID NO.7, SEQ ID NO.14, SEQ ID NO.24, SEQ ID NO.31, SEQ ID NO.40, SEQ ID NO.73, SEQ ID NO.74, SEQ ID NO.77, SEQ ID NO.78, SEQ ID NO.84, SEQ ID NO.96, SEQ ID NO.130, SEQ ID NO.134, SEQ ID NO.142 and SEQ ID NO. 153.

6. The gene chip of claim 5, wherein the DNA probes comprise 155 DNA probes, and the sequences of the DNA probes are shown in SEQ ID NO.1-SEQ ID NO. 155.

7. The gene chip according to claim 5 or 6, wherein the DNA probe is a biotin-labeled DNA probe.

8. A method of screening for an african swine fever virus genetic mutation, the method comprising:

1) extracting the genome DNA of a sample to be detected;

2) the DNA is broken to the range of 200-300 bp;

3) preparing a DNA fragment library from the fragmented genomic DNA;

4) hybridizing the DNA small fragment library with the sequence combination according to any one of claims 1 to 4 or the gene chip according to any one of claims 5 to 7, and detecting gene mutation.

9. Use of the sequence combination according to any one of claims 1 to 4 or the gene chip according to any one of claims 5 to 7 for the detection of African swine fever.