WO2020073810A1 - Method for detecting african swine fever virus - Google Patents

Abstract

Disclosed are a method and a primer pair for detecting African swine fever virus.

Description

African swine fever virus detection method Technical field

The invention relates to a method for detecting African swine fever virus (African Swine Fever Virus, ASFV), in particular to a method for detecting African swine fever virus (ASFV) using oligonucleotide pairs.

Background technique

Pigs are one of the most important economic animals in the world. According to statistics from the US Department of Agriculture, total pork production in the world's major pig-producing countries reached 113,081 thousand tons in 2018. The economic value of pork has exceeded 100 billion US dollars. However, intensive farming coupled with poor management may cause pigs to become susceptible to disease and the epidemic rapidly spreads. Take the African Swine Fever (ASF) outbreak in China in 2018 as an example. The mortality rate of sick pigs is almost 100%, which has caused huge economic losses. This shows that intensive monitoring of pig diseases in the farm is an indispensable part of management. The invention provides a rapid and convenient African swine fever virus (ASFV) detection method for industrial utilization.

Summary of the invention

In one aspect, the invention relates to an African swine fever virus (ASFV) detection method, comprising providing a sample that may contain one or more nucleotide sequences of an African swine fever virus (ASFV); providing an oligonucleotide Primer pair, the oligonucleotide primer pair includes a first primer and a second primer, the oligonucleotide primer pair is defined in one or more nucleotide sequences of the African swine fever virus (ASFV) 5 'end of the two complementary strands of the previous double strand target sequence; provide a polymerase; mix the sample, the oligonucleotide primer pair, the polymerase, and deoxyadenosine triphosphates in a container , dATPs), deoxycytidine triphosphates (dCTPs), deoxyguanosine triphosphates (dGTPs), and deoxythymidine triphosphates (dTTPs) to form a polymerase chain reaction (polymerase chain reaction, PCR) mixture; by heating the bottom of the container at a fixed temperature, the PCR mixture undergoes thermal convection polymerase chain reaction (convective polymerase chain reaction) reaction, cPCR) to form a PCR product; and detecting the PCR product to identify the double-stranded target sequence.

In certain embodiments, the sequence combination of the first primer and the second primer is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO : 5, SEQ ID NO: 7 and SEQ ID NO: 5, SEQ ID NO: 9 and SEQ ID NO: 11, SEQ ID NO: 9 and SEQ ID NO: 12, SEQ ID NO: 9 and SEQ ID NO: 13 , SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 10 and SEQ ID NO: 12, SEQ ID NO: 10 and SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 18, SEQ ID: NO: 15 and SEQ ID: NO: 19, SEQ ID: NO: 16 and SEQ ID: NO: 18, SEQ ID: NO: 16 and SEQ ID: NO: 19, SEQ ID: NO: 17 and SEQ ID: NO: 18, and SEQ ID NO: 17 and SEQ ID NO: 19.

In certain embodiments, the polymerase chain reaction (PCR) mixture further comprises an oligonucleotide probe comprising a segment complementary to a segment of the double-stranded target sequence Sequence, a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescent inhibitor molecule attached to a second position on the oligonucleotide probe, when When the oligonucleotide probe is not hybridized to the segment of the double-stranded target sequence, the fluorescent inhibitory molecule generally inhibits the fluorescent molecule, and when the oligonucleotide probe When hybridized to the segment of the double-stranded target sequence, the fluorescent molecule is not substantially inhibited.

In some embodiments, the sequence of the first primer is shown in SEQ ID NO: 1, and the sequence of the second primer is shown in SEQ ID NO: 2. In some embodiments, the polymerase chain reaction (PCR) mixture further comprises an oligonucleotide probe whose sequence is a complete p72 gene sequence (GenBank accession) between the African swine fever virus (ASFV) The oligonucleotide sequence of 13 to 30 base pairs between No. 1162 to No. 1206 nucleotides of No. MH713612). In certain preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 3.

In some embodiments, the sequence of the first primer is shown in SEQ ID NO: 4, and the sequence of the second primer is shown in SEQ ID NO: 5. In some embodiments, the polymerase chain reaction (PCR) mixture further comprises an oligonucleotide probe whose sequence is a complete p72 gene sequence (GenBank accession) between the African swine fever virus (ASFV) No. MH713612) The nucleotide sequence of 13 to 30 base pairs between the 454th to 503rd nucleotides. In certain preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 6.

In some embodiments, the sequence of the first primer is shown in SEQ ID NO: 7, and the sequence of the second primer is shown in SEQ ID NO: 5. In some embodiments, the polymerase chain reaction (PCR) mixture further comprises an oligonucleotide probe whose sequence is a complete p72 gene sequence (GenBank accession) between the African swine fever virus (ASFV) No. MH713612) 13 to 30 base pair oligonucleotide sequence between 454 to 583 nucleotides. In certain preferred embodiments, the sequence of the oligonucleotide probe is selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 8.

In certain embodiments, the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 9 and SEQ ID NO: 11, SEQ ID NO: 9 and SEQ ID NO : 13, SEQ ID NO: 10 and SEQ ID NO: 11, and SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the polymerase chain reaction (PCR) mixture further comprises an oligonucleotide probe whose sequence is a complete p72 gene sequence (GenBank accession) between the African swine fever virus (ASFV) The oligonucleotide sequence of 13 to 30 base pairs between No. MH713612) and No. MH713612). In certain preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 14.

In some embodiments, the sequence of the first primer is selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10, and the sequence of the second primer is shown in SEQ ID NO: 12. In some embodiments, the polymerase chain reaction (PCR) mixture further comprises an oligonucleotide probe whose sequence is a complete p72 gene sequence (GenBank accession) between the African swine fever virus (ASFV) The oligonucleotide sequence of 13 to 30 base pairs between the 403rd to 453rd nucleotides of No. MH713612). In certain preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 14.

In certain embodiments, the sequence combination of the first primer and the second primer is selected from the group comprising: SEQ ID NO: 15 and SEQ ID NO: 18, SEQ ID NO: 15 and SEQ ID NO : 19, SEQ ID NO: 16 and SEQ ID NO: 18, SEQ ID NO: 16 and SEQ ID NO: 19, SEQ ID NO: 17 and SEQ ID NO: 18, and SEQ ID NO: 17 and SEQ ID NO: 19. In some embodiments, the polymerase chain reaction (PCR) mixture further comprises an oligonucleotide probe whose sequence is a complete p72 gene sequence (GenBank accession) between the African swine fever virus (ASFV) No. MH713612) 13 to 30 base pair oligonucleotide sequence between 156th to 189th nucleotides. In certain preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 20.

In one aspect, the present invention relates to an oligonucleotide pair for detecting African swine fever virus (ASFV), which includes a first primer and a second primer.

In some embodiments, the sequence of the first primer is shown in SEQ ID NO: 1, and the sequence of the second primer is shown in SEQ ID NO: 2. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an entire p72 gene sequence between the African swine fever virus (ASFV) ( GenBank accession No. MH713612), an oligonucleotide sequence of 13 to 30 base pairs between 1162 to 1206 nucleotides, a first position attached to the oligonucleotide probe Fluorescent molecule, and a fluorescent inhibitory molecule attached to the oligonucleotide probe at a second position. In certain preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 3.

In some embodiments, the sequence of the first primer is shown in SEQ ID NO: 4, and the sequence of the second primer is shown in SEQ ID NO: 5. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an entire p72 gene sequence between the African swine fever virus (ASFV) ( GenBank accession No. MH713612), an nucleotide sequence of 13 to 30 base pairs between the 454th to 503rd nucleotides, a first position attached to the oligonucleotide probe Fluorescent molecule, and a fluorescent inhibitory molecule attached to the oligonucleotide probe at a second position. In certain preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 6.

In some embodiments, the sequence of the first primer is shown in SEQ ID NO: 7, and the sequence of the second primer is shown in SEQ ID NO: 5. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an entire p72 gene sequence between the African swine fever virus (ASFV) ( GenBank accession No. MH713612), an nucleotide sequence of 13 to 30 base pairs between the 454th to 583rd nucleotides, a first position attached to the oligonucleotide probe Fluorescent molecule, and a fluorescent inhibitory molecule attached to the oligonucleotide probe at a second position. In certain preferred embodiments, the sequence of the oligonucleotide probe is selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 8.

In certain embodiments, the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 9 and SEQ ID NO: 11, SEQ ID NO: 9 and SEQ ID NO : 13, SEQ ID NO: 10 and SEQ ID NO: 11, and SEQ ID NO: 10 and SEQ ID NO: 13. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an entire p72 gene sequence between the African swine fever virus (ASFV) ( GenBank Accession No. MH713612), an oligonucleotide sequence of 13 to 30 base pairs between the 403th to 442nd nucleotides, a first position attached to the oligonucleotide probe Fluorescent molecule, and a fluorescent inhibitory molecule attached to the oligonucleotide probe at a second position. In certain preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 14.

In some embodiments, the sequence of the first primer is selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10, and the sequence of the second primer is shown in SEQ ID NO: 12. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an entire p72 gene sequence between the African swine fever virus (ASFV) ( GenBank Accession No. MH713612), an oligonucleotide sequence of 13 to 30 base pairs between the 403th to 453rd nucleotides, a first position attached to the oligonucleotide probe Fluorescent molecule, and a fluorescent inhibitory molecule attached to the oligonucleotide probe at a second position. In certain preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 14.

In certain embodiments, the sequence combination of the first primer and the second primer is selected from the group comprising: SEQ ID NO: 15 and SEQ ID NO: 18, SEQ ID NO: 15 and SEQ ID NO : 19, SEQ ID NO: 16 and SEQ ID NO: 18, SEQ ID NO: 16 and SEQ ID NO: 19, SEQ ID NO: 17 and SEQ ID NO: 18, and SEQ ID NO: 17 and SEQ ID NO: 19. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an entire p72 gene sequence between the African swine fever virus (ASFV) ( GenBank Accession No. MH713612) 13 to 30 base pair oligonucleotide sequence between 156th to 189th nucleotides, a first position attached to the oligonucleotide probe Fluorescent molecule, and a fluorescent inhibitory molecule attached to the oligonucleotide probe at a second position. In certain preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 20.

All technical and scientific terms in this specification, unless otherwise defined, are in the field and have the meaning commonly understood by those skilled in the art.

The present invention is exemplified and illustrated by the following examples, but the present invention is not limited by the following examples.

BRIEF DESCRIPTION

Figure 1 shows the results of real-time PCR using primers ASFV-F1 and ASFV-R1 and probe ASFV-P1 to detect African swine fever virus (ASFV).

Figure 2 shows the results of real-time PCR using primers ASFV-F2 and ASFV-R2 and probe ASFV-P2 to detect African swine fever virus (ASFV).

detailed description

In one aspect, the invention relates to an African swine fever virus (ASFV) detection method. In some embodiments, the method is polymerase chain reaction (PCR). In certain embodiments, the method is reverse-transcription polymerase chain reaction (RT-PCR). In certain embodiments, the method is thermal convection polymerase chain reaction (cPCR). In some embodiments, the method is real-time polymerase chain reaction (real-time PCR).

In one aspect, the invention relates to an African swine fever virus (ASFV) detection method, comprising:

Provide a sample that may contain one or more nucleotide sequences of an African swine fever virus (ASFV);

An oligonucleotide primer pair is provided. The oligonucleotide primer pair includes a first primer and a second primer. The oligonucleotide primer pair is defined as one of the African swine fever virus (ASFV) or 5 'end of two complementary strands of a double strand target sequence on multiple nucleotide sequences; providing a polymerase;

Mix the sample, the oligonucleotide primer pair, the polymerase, deoxyadenosine triphosphate (dATPs), deoxycytidine triphosphate (dCTPs), deoxyguanosine triphosphate (dGTPs) in a container , And deoxythymidine triphosphates (dTTPs) to form a polymerase chain reaction (PCR) mixture;

By heating the bottom of the container at a fixed temperature, subjecting the PCR mixture to thermal convection polymerase chain reaction (cPCR) to form a PCR product; and

The PCR product is detected to identify the double-stranded target sequence.

In another aspect, the present invention relates to an African swine fever virus (ASFV) detection method, comprising:

Provide a sample that may contain one or more nucleotide sequences of an African swine fever virus (ASFV);

An oligonucleotide primer pair is provided. The oligonucleotide primer pair includes a first primer and a second primer. The oligonucleotide primer pair is defined as one of the African swine fever virus (ASFV) or 5 'ends of two complementary strands of a double strand target sequence on multiple nucleotide sequences;

An oligonucleotide probe is provided, the oligonucleotide probe comprising a sequence complementary to a segment of the double-stranded target sequence, a first attached to the oligonucleotide probe Fluorescent molecule at the position, and a fluorescent inhibitory molecule attached to the oligonucleotide probe at a second position, when the oligonucleotide probe is not hybridized to the double-stranded target sequence In the segment, the fluorescent inhibitory molecule generally inhibits the fluorescent molecule, and when the oligonucleotide probe is hybridized to the segment of the double-stranded target sequence, the fluorescent Photomolecules are generally not suppressed;

Provide a polymerase;

Mix the sample, the oligonucleotide primer pair, the oligonucleotide probe, the polymerase, deoxyadenosine triphosphate (dATPs), deoxycytidine triphosphate (dCTPs) in a container , Deoxyguanosine triphosphates (dGTPs), and deoxythymidine triphosphates (dTTPs) to form a polymerase chain reaction (PCR) mixture;

By heating the bottom of the container at a fixed temperature, subjecting the PCR mixture to thermal convection polymerase chain reaction (cPCR) to form a PCR product; and

The PCR product is detected to identify the double-stranded target sequence.

In yet another aspect, the present invention relates to an oligonucleotide pair for detecting African swine fever virus (ASFV).

In still another aspect, the present invention relates to an oligonucleotide pair and oligonucleotide probe for detecting African swine fever virus (ASFV)

It should be further understood that in certain embodiments, the oligonucleotide pairs and / or oligonucleotide probes disclosed herein can be used for variations in various basic PCR techniques, such as, but not limited to, reverse transcription polymerization Enzyme chain reaction (RT-PCR), thermal convection polymerase chain reaction (cPCR), real-time polymerase chain reaction (real-time PCR), nested polymerase chain reaction (nested PCR), and thermal asymmetry Sex interleaved polymerase chain reaction (thermal asymmetric interlaced PCR, TAIL-PCR).

As used herein, the term "thermal convection polymerase chain reaction (cPCR)" refers to a polymerase chain reaction in which the bottom of a tubular container containing a PCR sample is embedded in a stable heat source and the PCR is controlled The parameters, including the total volume, viscosity, surface temperature of the PCR sample, and the inner diameter of the tubular container, cause the temperature gradient from the bottom to the top of the PCR sample to decrease, induce thermal convection and cause the PCR sample to denature and adhere The polymerization occurs sequentially and repeatedly in different areas of the tubular container. For a detailed description of thermal convection polymerase chain reaction (cPCR), see, eg, US Patent No. 8,187,813, which is incorporated herein by reference in its entirety.

As used herein, the term "fluorescent molecule" means a substance or a portion thereof, which is capable of displaying fluorescence within a detectable range. As used herein, the term "fluorescence-inhibiting molecule" means a substance or a portion thereof that is capable of suppressing the fluorescence emitted by the fluorescent molecule when excited by a light source. In certain embodiments, the terms "fluorescent molecule" and "fluorescent inhibitory molecule" are fluorescent molecules and fluorescent inhibitory molecules of the TaqMan ^™ analysis kit (Applied Biosystems Inc., California, United States). For a detailed description of the TaqMan ^TM analysis kit, please see, for example, Holland et al., Proc. Natl. Acad. Sci, USA (1991) 88: 7276-7280; US Patent Nos. 5,538,848, 5,723,591, 5,876,930, and 7,413,708 are all cited. Way to incorporate its entirety into this article.

Examples of the fluorescent molecule include, but are not limited to, 3- (ε-carboxy) -3'-ethyl-5,5'-dimethylhexylcarbocyanine (3- (ε-carboxypentyl) -3 ' -ethyl-5,5'-dimethyloxa-carbocyanine (CYA), 6-carboxyfluorescein (FAM), 5,6-carboxyrhodamine-L LO (5,6-carboxyrhodamine-lOl, R110 ), 6-carboxyrhodamine-6G (6-carboxyrhodamine-6G, R6G), N ', N', N ', N'-tetramethyl-6-carboxyrhodamine (N', N ', N', N '-tetramethyl-6-carboxyrhodamine, TAMRA), 6-carboxy-X-rhodamine (6-carboxy-X-rhodamine, ROX), 2', 4 ', 5', 7'-tetrachloro 4-7-di Chlorofluorescein (2 ', 4', 5 ', 7'-tetrachloro-4-7-dichlorofluorescein, TET), 2', 7-dimethoxy-4 ', 5'-6 carboxyrhodamine (2 ', 7-dimethoxy-4', 5'-6carboxyrhodamine, JOE), 6-carboxy-2 ', 4,4', 5 ', 7,7'-hexachlorofluorescein (6-carboxy-2', 4 , 4 ', 5', 7,7'-hexachlorofluorescein, HEX), ALEXA fluorescent, Cy3 fluorescent and Cy5 fluorescent. Examples of the fluorescence inhibitory molecule include, but are not limited to, 4- (4'-dimethylamino-phenylazo) -benzoic acid (4- (4'-dimethylamino-phenylazo) -benzoic acid, Dabcyl), Black hole fluorescent inhibitor 1 (Black Hole Quencher 1, BHQ1), black hole fluorescent inhibitor 2 (Black Hole Quencher 2, BHQ2), black hole fluorescent inhibitor 3 (Black Hole Quencher 3, BHQ3), dihydrocyclopyrrole Indole tripeptide minor groove conjugate (dihydro cyclopyrole indole tripeptide mineral binder (MGB), tetramethylrhodamine (tetramethylrhodamine, TAMRA). In certain embodiments, the fluorescent molecule is 6-carboxyluciferin (FAM), and the fluorescent inhibitory molecule is dihydrocyclopyrroloindole tripeptide minor groove conjugate (MGB). In certain embodiments, the fluorescent molecule is 6-carboxyfluorescein (FAM), and the fluorescent inhibitory molecule is black hole fluorescent inhibitor 1 (BHQ1) or tetramethylrhodamine (TAMRA).

Unless otherwise defined herein, the scientific and technical terms used in conjunction with this document shall have the meaning commonly understood by those of ordinary skill in the art. In addition, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present invention can generally be carried out according to conventional methods known in the art. Generally speaking, the nomenclature described in this article to link the following technologies, as well as the technologies of biochemistry, enzymology, molecular and cell biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization reactions are all in the field Known and frequent users. Unless otherwise stated, the methods and techniques of the present invention can generally be performed according to conventional methods known in the art, and are described in various general and more specific references that are cited and discussed in this specification.

The invention is further illustrated by the following examples, which should not be interpreted as a further limitation in any way. The entire contents of all cited documents cited in this application (including references, approved patents, published patent applications, and patent applications filed together) are hereby expressly incorporated by reference.

Examples

Example 1 Detection of African swine fever virus (ASFV) by traditional polymerase chain reaction

The full length sequence of p72 gene (GenBank accession No. MH713612) of African swine fever virus (ASFV) is inserted into a cloning vector, which may be, but not limited to, pUC57, pGEM-T to obtain the pASFV plasmid.

The 50 μl PCR mix used for traditional PCR contains: 106 copy number pASFV plasmids, 0.01-2 μM forward primers, 0.01-2 μM reverse primers, 0.2 μM dNTPs and 1.25 U Taq DNA polymerase. Perform the amplification reaction in a thermal cycler (eg, but not limited to PC818, Astec Co. Ltd., Japan), and include an initial cycle of denaturation at 94 ° C for 3 minutes, and 35 cycles of 94 ° C for 30 seconds, 30 seconds at 60 ° C and 30 seconds at 72 ° C. The amplified product was then analyzed with 15% polyacrylamide gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA), and visualized by staining with ethidium bromide.

The results of traditional PCR are shown in Table 1. Each primer pair amplified the correct size fragment of each target sequence, while no target sequence was amplified in the negative control group (results not shown). The results indicate that each primer pair can be used in traditional PCR amplification reactions to detect the presence of African swine fever virus (ASFV).

Table 1 The results of traditional PCR for each primer pair

Forward primer	Reverse primer	Synthetic fragment size (bp)
ASFV-F1 (SEQ ID NO: 1)	ASFV-R1 (SEQ ID NO: 2)	87
ASFV-F2 (SEQ ID NO: 4)	ASFV-R2 (SEQ ID NO: 5)	92
ASFV-F3 (SEQ ID NO: 7)	ASFV-R2 (SEQ ID NO: 5)	174
ASFV-F4 (SEQ ID NO: 9)	ASFV-R4 (SEQ ID NO: 11)	81
ASFV-F5 (SEQ ID NO: 10)	ASFV-R4 (SEQ ID NO: 11)	84
ASFV-F4 (SEQ ID NO: 9)	ASFV-R5 (SEQ ID NO: 12)	90
ASFV-F5 (SEQ ID NO: 10)	ASFV-R5 (SEQ ID NO: 12)	93
ASFV-F4 (SEQ ID NO: 9)	ASFV-R6 (SEQ ID NO: 13)	81
ASFV-F5 (SEQ ID NO: 10)	ASFV-R6 (SEQ ID NO: 13)	84
ASFV-dF7 (SEQ ID NO: 15)	ASFV-dR7 (SEQ ID NO: 18)	81
ASFV-dF7 (SEQ ID NO: 15)	ASFV-dR8 (SEQ ID NO: 19)	82
ASFV-dF8 (SEQ ID NO: 16)	ASFV-dR7 (SEQ ID NO: 18)	82

Forward primer	Reverse primer	Synthetic fragment size (bp)
ASFV-dF8 (SEQ ID NO: 16)	ASFV-dR8 (SEQ ID NO: 19)	83
ASFV-dF9 (SEQ ID NO: 17)	ASFV-dR7 (SEQ ID NO: 18)	81
ASFV-dF9 (SEQ ID NO: 17)	ASFV-dR8 (SEQ ID NO: 19)	82

Example 2 Detection of African swine fever virus (ASFV) by thermal convection polymerase chain reaction (cPCR)

The 50 μl PCR mixture used for thermal convection polymerase chain reaction (cPCR) contains: a plasmid with the full-length sequence of the p72 gene of African swine fever virus (ASFV) (pASFV plasmid, same as in Example 1) (102, 103, 104, 105, 106 copy numbers, respectively) ), 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.01-2μM probe (the 5 'end of each probe sequence is joined to a fluorescent molecule 6-carboxyfluorescein (FAM), and each probe sequence The 3 'end is joined with a fluorescent inhibitor molecule black hole fluorescent inhibitor 1 (BHQ1) or tetramethylrhodamine (TAMRA)), 0.2 μM dNTP, 1X PCR buffer, and 1-5U Taq DNA polymerase. The PCR mixture is added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) apparatus for a specified period of time (about 30 to 45 minutes). The cPCR instrument was used to detect the FAM fluorescence in each sample. The above cPCR analysis test was repeated 8 times (n = 8) to evaluate the sensitivity of each estimated primer to the probe.

The results of the sensitivity test are shown in Table 2. Each primer pair and probe combination can 100% correctly detect the pASFV plasmid containing 102 copies of the sample, and the sensitivity can reach 102 copies.

Table 2 Sensitivity test results of each primer pair and probe combination (n = 8)

In addition, the specificity of each of the above primer pairs and probe combinations was analyzed using different swine pathogen gene plasmids (106 copies / μl) as cPCR templates. The cPCR method is as described above. The results of the specificity test are shown in Table 3. Each primer pair and probe combination can correctly detect the sample containing African swine fever virus (ASFV), but cannot detect the sample containing classical swine fever virus (classical swine fever virus). Fever virus (CSFV), foot and mouth disease virus (Foot and mouth disease virus (FMDV), Japanese encephalitis virus (Japanese Encephalitis Virus, JEV), porcine reproductive and respiratory syndrome virus (Porcine Reproductive virus and PRRSV), type A pig Influenza virus (SIV), Porcine epidemial virus (PEDV), Porcine circovirus type 2, PCV2, Pseudorabies virus (PRV) sample. The results show that each primer pair and probe combination are specific.

Table 3 Specific test results of each primer pair and probe combination

"+" Means that a fluorescent signal was detected in the sample, and "-" means that no fluorescent signal was detected in the sample.

Example 3 Detection of African swine fever virus (ASFV) by real-time quantitative polymerase chain reaction (real-time PCR, qPCR)

Real-time quantitative PCR analysis was performed in a real-time quantitative PCR instrument (eg, but not limited to ABI StepOnePlusTM; Applied BioSystem, Life Technologies, California, United States) with diluted pASFV plasmids (100, 101, 102, 103, 104, 105, 106, and 107 copy numbers, respectively). It contains 2μl pASFV plasmid, 0.01-2μM forward primer ASFV-F1 (SEQ ID NO: 1), 0.01-2μM reverse primer ASFV-R1 (SEQ ID NO: 2), 0.01-2μM probe ASFV-P1 (5 'FAM-CACAAGCCGCACCAAAGCAAACCT-BHQ1'3', SEQ ID NO: 3) Commercial RT-PCR kit with a total volume of 20 μl (for example, but not limited to, OneStep PrimeScriptTM RT-PCR Kit; Takara Bio Inc., Japan) for real-time quantification PCR analysis. The real-time quantitative PCR program is 42 ° C for 5 minutes, 94 ° C for 10 seconds, and 40 cycles of 94 ° C for 10 seconds and 60 ° C for 30 minutes. Record the results of the fluorescence measurement in a 60 ° C step.

As shown in FIG. 1, a standard curve for real-time quantitative PCR analysis of pASFV plasmids serially diluted (10-fold) was calculated. One copy number of pASFV plasmid can be detected. The R2 value of the standard curve is 0.99812, which indicates that the primer pair and the probe of the present invention can be used for real-time quantitative PCR and produce reliable results.

In addition, another set of primer pairs and probes were used for real-time quantitative PCR analysis. It contains 2μl of pASFV plasmid (100, 101, 102, 103, 104, 105, 106, 107 copies), 0.01-2μM forward primer ASFV-F2 (SEQ ID NO: 4), 0.01-2μM reverse primer ASFV-R2 (SEQ ID NO: 5), 0.01-2μM probe ASFV-P2 (5'FAM-TCCTCATCAACACCGAGATTGGCACA-BHQ1'3 ', SEQ ID NO: 6) commercial RT-PCR kit with a total volume of 20μl (for example, but not limited to, OneStep PrimeScriptTM RT-PCR Kit; Takara Bio Inc., Japan) for real-time quantitative PCR analysis. The real-time quantitative PCR program is 42 ° C for 5 minutes, 94 ° C for 10 seconds, and 40 cycles of 94 ° C for 10 seconds and 60 ° C for 30 minutes. Record the results of the fluorescence measurement in a 60 ° C step.

As shown in Fig. 2, a standard curve for real-time quantitative PCR analysis of pASFV plasmids serially diluted (10-fold) was calculated. At least 10 copies of pASFV plasmid can be detected. The R2 value of the standard curve is 0.9998, which indicates that the primer pair and the probe of the present invention can be used for real-time quantitative PCR and produce reliable results.

The results of Examples 1-3 show that the primer pairs and probes of the present invention can be used for different polymerase chain reaction, including traditional PCR, cPCR, qPCR, etc., to detect the presence of African swine fever virus (ASFV) Highly sensitive and specific.

The above detailed description is a specific description of a feasible embodiment of the present invention, but this embodiment is not intended to limit the patent scope of the present invention, and any equivalent implementation or change without departing from the technical spirit of the present invention should include In the patent scope of this case.

Claims (13)

1. An oligonucleotide pair for detecting African swine fever virus, comprising a first primer and a second primer, the sequence combination of the first primer and the second primer is selected from the group consisting of : SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 5, SEQ ID NO: 9 and SEQ ID NO: 11, SEQ ID: NO: 9 and SEQ ID: NO: 12, SEQ ID: NO: 9 and SEQ ID: NO: 13, SEQ ID: NO: 10 and SEQ ID: NO: 11, SEQ ID: NO: 10 and SEQ ID: NO: 12, SEQ ID: NO : 10 and SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 18, SEQ ID NO: 15 and SEQ ID NO: 19, SEQ ID NO: 16 and SEQ ID NO: 18, SEQ ID NO: 16 And SEQ ID NO: 19, SEQ ID NO: 17 and SEQ ID NO: 18, and SEQ ID NO: 17 and SEQ ID NO: 19.
2. The oligonucleotide pair of claim 1, wherein the sequence combination of the first primer and the second primer is SEQ ID NO: 1 and SEQ ID NO: 2, and further includes an oligonucleotide probe Needle, the oligonucleotide probe comprises an oligonucleotide sequence of 13 to 30 base pairs between the 1162th to 1206th nucleotides of the p72 gene sequence of the African swine fever virus, A fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescent inhibitor molecule attached to a second position on the oligonucleotide probe.
3. The oligonucleotide pair according to claim 2, wherein the sequence of the oligonucleotide probe is shown in SEQ ID NO: 3.
4. The oligonucleotide pair of claim 1, wherein the sequence combination of the first primer and the second primer is SEQ ID NO: 4 and SEQ ID NO: 5, and further comprises an oligonucleotide probe Needle, the oligonucleotide probe comprises an oligonucleotide sequence of 13 to 30 base pairs between the 454th to 503rd nucleotides of the p72 gene sequence of the African swine fever virus, A fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescent inhibitor molecule attached to a second position on the oligonucleotide probe.
5. The oligonucleotide pair according to claim 4, wherein the sequence of the oligonucleotide probe is shown in SEQ ID NO: 6.
6. The oligonucleotide pair of claim 1, wherein the sequence combination of the first primer and the second primer is SEQ ID NO: 7 and SEQ ID NO: 5, and further comprises an oligonucleotide probe Needle, the oligonucleotide probe comprises an oligonucleotide sequence of 13 to 30 base pairs between the 454th to 583rd nucleotides of the p72 gene sequence of the African swine fever virus, A fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescent inhibitor molecule attached to a second position on the oligonucleotide probe.
7. The oligonucleotide pair of claim 6, wherein the sequence of the oligonucleotide probe is selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 8.
8. The oligonucleotide pair of claim 1, wherein the sequence combination of the first primer and the second primer is selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 11, SEQ ID: NO: 9 and SEQ ID: NO: 13, SEQ ID: NO: 10 and SEQ ID: NO: 11, and SEQ ID: NO: 10 and SEQ ID: NO: 13, and further comprising an oligonucleotide probe, the oligo The nucleotide probe includes an oligonucleotide sequence of 13 to 30 base pairs between the 403th to 442th nucleotides of the p72 gene sequence of the African swine fever virus, an additional A fluorescent molecule at a first position on the oligonucleotide probe, and a fluorescent inhibitor molecule at a second position attached to the oligonucleotide probe.
9. The oligonucleotide pair according to claim 8, wherein the sequence of the oligonucleotide probe is shown in SEQ ID NO: 14.
10. The oligonucleotide pair of claim 1, wherein the sequence combination of the first primer and the second primer is selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 12, and SEQ ID NO: 10 and SEQ ID NO: 12, and further includes an oligonucleotide probe, the oligonucleotide probe includes a 403th to 453rd of the p72 gene sequence of the African swine fever virus An oligonucleotide sequence of 13 to 30 base pairs between nucleotides, a fluorescent molecule attached to a first position on the oligonucleotide probe, and an oligonucleotide attached to the oligo A second position of fluorescence inhibitor on the nucleotide probe.
11. The oligonucleotide pair according to claim 10, wherein the sequence of the oligonucleotide probe is shown in SEQ ID NO: 14.
12. The oligonucleotide pair of claim 1, wherein the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 15 and SEQ ID NO: 18, SEQ ID: NO: 15 and SEQ ID: NO: 19, SEQ ID: NO: 16 and SEQ ID: NO: 18, SEQ ID: NO: 16 and SEQ ID: NO: 19, SEQ ID: NO: 17 and SEQ ID: NO: 18, and SEQ ID NO: 17 and SEQ ID NO: 19, and further comprising an oligonucleotide probe comprising an 156th to 189th nucleus between the p72 gene sequence of the African swine fever virus An oligonucleotide sequence of 13 to 30 base pairs between nucleotides, a fluorescent molecule attached to a first position on the oligonucleotide probe, and an oligonucleotide attached to the oligonucleotide A fluorescent inhibitory molecule at a second position on the acid probe.
13. The oligonucleotide pair according to claim 12, wherein the sequence of the oligonucleotide probe is shown in SEQ ID NO: 20.

Images