TWI731425B - Pairs of oligonucleotides and probes for detecting african swine fever virus (asfv) - Google Patents

Abstract

The present invention relates to a method for detecting African swine fever virus (ASFV). In addition, the present invention also relates to pairs of oligonucleotides for detecting African swine fever virus (ASFV).

Description

An oligonucleotide pair and probe for detecting African swine fever virus

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

Pigs are one of the most important economic animals in the world. According to statistics from the U.S. Department of Agriculture, the total pork production of the world's major pig raising countries in 2018 reached 113,081 thousand tons. The economic value of pork has exceeded 100 billion U.S. dollars. However, intensive breeding combined with poor management may make pigs susceptible to disease and rapid spread of the epidemic. Taking 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. It can be seen that intensive monitoring of pig diseases in the feedlot is an indispensable part of management. The invention provides a fast and convenient method for detecting African swine fever virus (ASFV) for industrial use.

In one aspect, the present 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 Yes, the oligonucleotide primer pair includes a first primer and a second primer, the 5'end of the two complementary strands of the oligonucleotide row; a polymerase is provided; the sample and the oligonucleotide are mixed in a container Acid primer pairs, the polymerase, deoxyadenosine triphosphates (dATPs), deoxycytidine triphosphates (dCTPs), 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, the PCR mixture is subjected to thermal convective polymerase chaining Reaction (convective polymerase chain reaction, cPCR) to form a PCR product; and detecting the PCR product to identify the double-stranded target sequence.

In some specific embodiments, the sequence combination of the first primer and the second primer is selected from the group comprising: 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 some embodiments, the polymerase chain reaction (PCR) mixture further includes an oligonucleotide probe, the oligonucleotide probe including a sequence complementary to a segment of the double-stranded target sequence, A fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescent molecule attached to a second position on the oligonucleotide probe, when the oligonucleotide When the probe is not hybridized to the region of the double-stranded target sequence, the fluorescence-inhibiting molecule substantially inhibits the fluorescent molecule, and when the oligonucleotide probe is hybridized to the region of the double-stranded target sequence For a while, the fluorescent molecule is not substantially suppressed.

In some specific 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 includes an oligonucleotide probe whose sequence is a complete p72 gene sequence (GenBank accession No. of the African swine fever virus (ASFV)). The oligonucleotide sequence of 13 to 30 base pairs between the 1162th and 1206th nucleotides of MH713612). In some preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 3.

In some specific 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 includes an oligonucleotide probe whose sequence is a complete p72 gene sequence (GenBank accession No. of the African swine fever virus (ASFV)). The oligonucleotide sequence of 13 to 30 base pairs between the 454th to 503rd nucleotides of MH713612). In some preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 6.

In some specific 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 includes an oligonucleotide probe whose sequence is a complete p72 gene sequence (GenBank accession No. of the African swine fever virus (ASFV)). The oligonucleotide sequence of 13 to 30 base pairs between the 454th to the 583th nucleotides of MH713612). In some 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 some specific embodiments, the sequence combination of the first primer and the second primer is selected from the group comprising: 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 includes an oligonucleotide probe whose sequence is a complete p72 gene sequence (GenBank accession No. of the African swine fever virus (ASFV)). The oligonucleotide sequence of 13 to 30 base pairs between the 403th to the 442th nucleotides of MH713612). In some 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 includes an oligonucleotide probe whose sequence is a complete p72 gene sequence (GenBank accession No. of the African swine fever virus (ASFV)). The oligonucleotide sequence of 13 to 30 base pairs between the 403th to the 453rd nucleotides of MH713612). In some preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 14.

In some specific 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 includes an oligonucleotide probe whose sequence is a complete p72 gene sequence (GenBank accession No. of the African swine fever virus (ASFV)). The oligonucleotide sequence of 13 to 30 base pairs between the 156th to 189th nucleotides of MH713612). In some 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), comprising a first primer and a second primer.

In some specific 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 oligonucleotide pair further includes an oligonucleotide probe, and the oligonucleotide probe includes an entire p72 gene sequence (GenBank accession) of the African swine fever virus (ASFV). No. MH713612) of the oligonucleotide sequence of 13 to 30 base pairs between the 1162th and 1206th nucleotides, a fluorescent light attached to a first position on the oligonucleotide probe Molecule, and a fluorescence-suppressing molecule attached to a second position on the oligonucleotide probe. In some preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 3.

In some specific 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 oligonucleotide pair further includes an oligonucleotide probe, and the oligonucleotide probe includes an entire p72 gene sequence (GenBank accession) of the African swine fever virus (ASFV). No. MH713612) the oligonucleotide sequence of 13 to 30 base pairs between the 454th to the 503rd nucleotide, a fluorescent light attached to a first position on the oligonucleotide probe Molecule, and a fluorescence-suppressing molecule attached to a second position on the oligonucleotide probe. In some preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 6.

In some specific 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 oligonucleotide pair further includes an oligonucleotide probe, and the oligonucleotide probe includes an entire p72 gene sequence (GenBank accession) of the African swine fever virus (ASFV). No. MH713612) of the oligonucleotide sequence of 13 to 30 base pairs between the 454th to the 583th nucleotide, a fluorescent light attached to a first position on the oligonucleotide probe Molecule, and a fluorescence-suppressing molecule attached to a second position on the oligonucleotide probe. In some 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 some specific embodiments, the sequence combination of the first primer and the second primer is selected from the group comprising: 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 oligonucleotide pair further includes an oligonucleotide probe, and the oligonucleotide probe includes an entire p72 gene sequence (GenBank accession) of the African swine fever virus (ASFV). No. MH713612) of the oligonucleotide sequence of 13 to 30 base pairs between the 403th to the 442nd nucleotides, a fluorescent light attached to a first position on the oligonucleotide probe Molecule, and a fluorescence-suppressing molecule attached to a second position on the oligonucleotide probe. In some 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 oligonucleotide pair further includes an oligonucleotide probe, and the oligonucleotide probe includes an entire p72 gene sequence (GenBank accession) of the African swine fever virus (ASFV). No. MH713612) of the oligonucleotide sequence of 13 to 30 base pairs between the 403 to 453 nucleotides, a fluorescent light attached to a first position on the oligonucleotide probe Molecule, and a fluorescence-suppressing molecule attached to a second position on the oligonucleotide probe. In some preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 14.

In some specific 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 oligonucleotide pair further includes an oligonucleotide probe, and the oligonucleotide probe includes an entire p72 gene sequence (GenBank accession) of the African swine fever virus (ASFV). No. MH713612) oligonucleotide sequence of 13 to 30 base pairs between the 156th and 189th nucleotides, a fluorescent light attached to a first position on the oligonucleotide probe Molecule, and a fluorescence-suppressing molecule attached to a second position on the oligonucleotide probe. In some preferred embodiments, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 20.

In one aspect, the present invention relates to a method for detecting African swine fever virus (ASFV). In some embodiments, the method is polymerase chain reaction (PCR). In some embodiments, the method is reverse-transcription polymerase chain reaction (RT-PCR). In some embodiments, the method is a convective polymerase chain reaction (cPCR). In some embodiments, the method is real-time polymerase chain reaction (real-time PCR).

In one aspect, the present invention relates to a method for detecting African swine fever virus (ASFV), 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, and the oligonucleotide primer pair is defined in one or more nuclei of the African swine fever virus (ASFV) The 5'end of the two complementary strands of a double-strand target sequence on the nucleotide sequence; Provide a polymerase; Mix the sample, the oligonucleotide primer pair, the polymerase, deoxyadenosine triphosphates (dATPs), deoxycytidine triphosphates (dCTPs), and deoxyguanosine triphosphates (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, the PCR mixture is subjected to a thermal convective 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 a method for detecting African swine fever virus (ASFV), 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, and the oligonucleotide primer pair is defined in one or more nuclei of the African swine fever virus (ASFV) The 5'end of the two complementary strands of a double-strand target sequence on the nucleotide sequence; An oligonucleotide probe is provided, the oligonucleotide probe comprising a sequence complementary to a segment of the double-stranded target sequence, a fluorescent light attached to a first position on the oligonucleotide probe Light molecule, and a fluorescence inhibitor attached to a second position on the oligonucleotide probe, when the oligonucleotide probe is not hybridized to the segment of the double-stranded target sequence, the The fluorescence-suppressing molecule substantially inhibits the fluorescent molecule, and when the oligonucleotide probe hybridizes to the segment of the double-stranded target sequence, the fluorescent molecule is substantially uninhibited; Provide a polymerase; Mix the sample, the oligonucleotide primer pair, the oligonucleotide probe, the polymerase, deoxyadenosine triphosphates (dATPs), deoxycytidine triphosphates (dCTPs), and deoxycytidine triphosphates (dCTPs) in a container. Oxyguanosine 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, the PCR mixture is subjected to a thermal convective 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 another aspect, the present invention relates to an oligonucleotide pair and an oligonucleotide probe for detecting African swine fever virus (ASFV).

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

As used herein, the term "thermal convective 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 parameters of the PCR are controlled, including The total volume, viscosity, surface temperature of the PCR sample, and the inner diameter of the tubular container make the temperature gradient from the bottom to the top of the PCR sample drop, induce thermal convection and cause the denaturation, adhesion, and polymerization of the PCR sample in the tubular container Occurs sequentially and repetitively in different areas of. For a detailed description of the thermal convective polymerase chain reaction (cPCR), please see, for example, 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 part thereof, which can display fluorescence within a detectable range. As used herein, the term "fluorescence-inhibiting molecule" means a substance or a part thereof, which is capable of suppressing the fluorescence emitted by the fluorescent molecule when excited by a light source. In some embodiments, the terms "fluorescent molecule" and "fluorescence-inhibiting molecule" refer to the fluorescent molecules and fluorescence-inhibiting molecules of the TaqMan™ analysis kit (Applied Biosystems Inc., California, USA). For a detailed description of the TaqMan™ analysis kit, please refer to, for example, Holland et al., Proc. Natl. Acad. Sci, USA (1991) 88:7276--7280; U.S. Patent Nos. 5,538,848, 5,723,591, 5,876,930, and 7,413,708 are all cited by reference Way to incorporate it as a whole into this article.

Examples of such fluorescent molecules include, but are not limited to, 3-(ε-carboxy)-3'-ethyl-5,5'-dimethylhexacarbocyanine (3-(ε-carboxypentyl)-3'- ethyl-5,5'-dimethyloxa-carbocyanine, CYA), 6-carboxyfluorescein (FAM), 5,6-carboxyrhodamine-L LO (5,6-carboxyrhodamine-l lO, 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'-tetrachloro4-7-dichloro Luciferin (2',4',5',7'-tetrachloro-4-7-dichlorofluorescein, TET), 2',7-dimethoxy-4',5'-6 carboxyrhodamine (2',7-dimethoxy-4',5'-6 carboxyrhodamine, JOE), 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (6-carboxy-2',4 ,4',5',7,7'-hexachlorofluorescein, HEX), ALEXA fluorescence, Cy3 fluorescence and Cy5 fluorescence. Examples of the fluorescence-inhibiting molecules include, but are not limited to, 4-(4'-dimethylamino-phenylazo)-benzoic acid (Dabcyl), black hole Fluorescence inhibitor 1 (Black Hole Quencher 1, BHQ1), black hole fluorescence inhibitor 2 (Black Hole Quencher 2, BHQ2), black hole fluorescence inhibitor 3 (Black Hole Quencher 3, BHQ3), dihydrocyclopyrroloindole Dole tripeptide minor groove binder (dihydro cyclo pyrrolo indole tripeptide minor groove binder, MGB), tetramethylrhodamine (tetramethylrhodamine, TAMRA). In some embodiments, the fluorescent molecule is 6-carboxyfluorescein (FAM), and the fluorescence-inhibiting molecule is dihydrocyclopyrroloindole tripeptide minor groove binder (MGB). In some embodiments, the fluorescent molecule is 6-carboxyfluorescein (FAM), and the fluorescence-inhibiting molecule is black hole fluorescence inhibitor 1 (BHQ1).

Unless otherwise defined herein, the scientific and technical terms used in conjunction with this document shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, 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 used to link the following technologies described herein, as well as the technologies of biochemistry, enzymes, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybrid reactions, are all in the field Known and frequent users. Unless otherwise specified, 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 cited and discussed in this specification.

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

Example

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

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

Conventional PCR used for 50 μl PCR mixture contained: pASFV plasmid copy number ^106, 0.01-2 μM forward primer, 0.01-2 μM reverse primer, 0.2 μM dNTP and 1.25U Taq DNA polymerase. In a thermal cycler (e.g., but not limited to PC818, Astec Co. Ltd., Japan) in the amplification reaction, and comprising an initial denaturation cycle a 94 ^o C for 3 minutes, and 35 cycles of 94 ^o C 30 Seconds, 60 ^o C for 30 seconds, and 72 ^o C for 30 seconds. The amplified products were then analyzed on 15% polyacrylamide gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) and stained with ethidium bromide appear.

The results of traditional PCR are shown in Table 1. Each primer pair amplified fragments of the correct size of each target sequence, but no target sequence was amplified in the negative control group (results not shown). This result shows that each primer pair can be used in the traditional PCR amplification reaction to detect the presence of African swine fever virus (ASFV). [Table 1] Results of traditional PCR for each primer pair Forward primer Back 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 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 Thermal convective polymerase chain reaction (cPCR) Detection of African swine fever virus (ASFV)

The 50 µl PCR mixture used for thermal convective polymerase chain reaction (cPCR) contains: plastids with the full-length sequence of the p72 gene of African swine fever virus (ASFV) (pASFV plastids, same as in Example 1) (10 ^{2 respectively)} , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copies), 0.01-2 µM forward primer, 0.01-2 µM reverse primer, 0.01-2 µM probe (the 5'end of each probe sequence is joined with a fluorescent Light molecule 6-carboxy fluorescein (FAM), and the 3'end of each probe sequence is connected with a fluorescence inhibitor black hole fluorescence inhibitor 1 (BHQ1) or tetramethylrhodamine (TAMRA)), 0.2 µM dNTP , 1X cPCR buffer, and 1-5 U Taq DNA polymerase. Add the PCR mixture to a reaction tube and place it in a thermal convective polymerase chain reaction (cPCR) instrument for a specified period of time (about 30 to 45 minutes). The cPCR machine was used to detect the FAM fluorescence in each sample. Repeat the above cPCR analysis test 8 times (n = 8) to evaluate the sensitivity of each primer pair and probe.

The results of the sensitivity tests shown in Table 2, each of the primer and probe combination Jieke 100% correct detected pASFV plastids contained in the sample copy number of ^102, a sensitivity of up to ¹⁰² copy numbers. [Table 2] Sensitivity test results of each primer pair and probe combination (n = 8) The positive rate of primer pair and probe combination% Negative control group pASFV plastid content (copy number) 10 ² 10 ³ 10 ⁴ 10 ⁵ 10 ⁶ ASFV-F1 (SEQ ID NO: 1) ASFV-R1 (SEQ ID NO: 2) ASFV-P1 (SEQ ID NO: 3) 0 100 100 100 100 100 ASFV-F2 (SEQ ID NO: 4) ASFV-R2 (SEQ ID NO: 5) ASFV-P2 (SEQ ID NO: 6) 0 100 100 100 100 100 ASFV-F3 (SEQ ID NO: 7) ASFV-R2 (SEQ ID NO: 5) ASFV-P3 (SEQ ID NO: 8) 0 100 100 100 100 100 ASFV-F4 (SEQ ID NO: 9) ASFV-R4 (SEQ ID NO: 11) ASFV-P4 (SEQ ID NO: 14) 0 100 100 100 100 100 ASFV-F5 (SEQ ID NO: 10) ASFV-R4 (SEQ ID NO: 11) ASFV-P4 (SEQ ID NO: 14) 0 100 100 100 100 100 ASFV-F4 (SEQ ID NO: 9) ASFV-R5 (SEQ ID NO: 12) ASFV-P4 (SEQ ID NO: 14) 0 100 100 100 100 100 ASFV-F5 (SEQ ID NO: 10) ASFV-R5 (SEQ ID NO: 12) ASFV-P4 (SEQ ID NO: 14) 0 100 100 100 100 100 ASFV-F4 (SEQ ID NO: 9) ASFV-R6 (SEQ ID NO: 13) ASFV-P4 (SEQ ID NO: 14) 0 100 100 100 100 100 ASFV-F5 (SEQ ID NO: 10) ASFV-R6 (SEQ ID NO: 13) ASFV-P4 (SEQ ID NO: 14) 0 100 100 100 100 100 ASFV-dF7 (SEQ ID NO: 15) ASFV-dR7 (SEQ ID NO: 18) ASFV-P7 (SEQ ID NO: 20) 0 100 100 100 100 100 ASFV-dF7 (SEQ ID NO: 15) ASFV-dR8 (SEQ ID NO: 19) ASFV-P7 (SEQ ID NO: 20) 0 100 100 100 100 100 ASFV-dF8 (SEQ ID NO: 16) ASFV-dR7 (SEQ ID NO: 18) ASFV-P7 (SEQ ID NO: 20) 0 100 100 100 100 100 ASFV-dF8 (SEQ ID NO: 16) ASFV-dR8 (SEQ ID NO: 19) ASFV-P7 (SEQ ID NO: 20) 0 100 100 100 100 100 ASFV-dF9 (SEQ ID NO: 17) ASFV-dR7 (SEQ ID NO: 18) ASFV-P7 (SEQ ID NO: 20) 0 100 100 100 100 100 ASFV-dF9 (SEQ ID NO: 17) ASFV-dR8 (SEQ ID NO: 19) ASFV-P7 (SEQ ID NO: 20) 0 100 100 100 100 100

In addition, different porcine pathogens plastid gene ⁽¹⁰⁶ copy number / μl) as a template cPCR analysis specific to each primer and probe combinations. 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 samples containing African swine fever virus (ASFV), but cannot detect classical swine fever virus (classical swine fever). virus, CSFV), Foot and mouth disease virus (FMDV), Japanese Encephalitis Virus (JEV), Porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza A Virus (Swine influenza A virus, SIV), Porcine epidemic diarrhea virus (PEDV), Porcine circovirus type 2, PCV2, Pseudorabies virus (PRV) sample. The results show that each primer pair and probe combination has specificity. [Table 3] Specificity test results of each primer pair and probe combination Combination of primer pair and probe ASFV CSFV FMDV JEV PRRSV SIV PEDV PCV2 PRV ASFV-F1 (SEQ ID NO: 1) ASFV-R1 (SEQ ID NO: 2) ASFV-P1 (SEQ ID NO: 3) + — — — — — — — — ASFV-F2 (SEQ ID NO: 4) ASFV-R2 (SEQ ID NO: 5) ASFV-P2 (SEQ ID NO: 6) + — — — — — — — — ASFV-F3 (SEQ ID NO: 7) ASFV-R2 (SEQ ID NO: 5) ASFV-P3 (SEQ ID NO: 8) + — — — — — — — — ASFV-F4 (SEQ ID NO: 9) ASFV-R4 (SEQ ID NO: 11) ASFV-P4 (SEQ ID NO: 14) + — — — — — — — — ASFV-F5 (SEQ ID NO: 10) ASFV-R4 (SEQ ID NO: 11) ASFV-P4 (SEQ ID NO: 14) + — — — — — — — — ASFV-F4 (SEQ ID NO: 9) ASFV-R5 (SEQ ID NO: 12) ASFV-P4 (SEQ ID NO: 14) + — — — — — — — — ASFV-F5 (SEQ ID NO: 10) ASFV-R5 (SEQ ID NO: 12) ASFV-P4 (SEQ ID NO: 14) + — — — — — — — — ASFV-F4 (SEQ ID NO: 9) ASFV-R6 (SEQ ID NO: 13) ASFV-P4 (SEQ ID NO: 14) + — — — — — — — — ASFV-F5 (SEQ ID NO: 10) ASFV-R6 (SEQ ID NO: 13) ASFV-P4 (SEQ ID NO: 14) + — — — — — — — — ASFV-dF7 (SEQ ID NO: 15) ASFV-dR7 (SEQ ID NO: 18) ASFV-P7 (SEQ ID NO: 20) + — — — — — — — — ASFV-dF7 (SEQ ID NO: 15) ASFV-dR8 (SEQ ID NO: 19) ASFV-P7 (SEQ ID NO: 20) + — — — — — — — — ASFV-dF8 (SEQ ID NO: 16) ASFV-dR7 (SEQ ID NO: 18) ASFV-P7 (SEQ ID NO: 20) + — — — — — — — — ASFV-dF8 (SEQ ID NO: 16) ASFV-dR8 (SEQ ID NO: 19) ASFV-P7 (SEQ ID NO: 20) + — — — — — — — — ASFV-dF9 (SEQ ID NO: 17) ASFV-dR7 (SEQ ID NO: 18) ASFV-P7 (SEQ ID NO: 20) + — — — — — — — — ASFV-dF9 (SEQ ID NO: 17) ASFV-dR8 (SEQ ID NO: 19) ASFV-P7 (SEQ ID NO: 20) + — — — — — — — — "+" means that a fluorescent signal is detected in the sample, and "-" means that no fluorescent signal is detected in the sample.

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

In diluted pASFV plastids (respectively ^{100, 101, 102, 103, 104, 105, 106, 107} copy numbers) in a real time PCR instrument (e.g., but not limited to ABI StepOnePlus ^TM ; Applied BioSystem, Life Technologies, California, USA) for real-time PCR analysis. With 2 µl pASFV plastid, 0.01-2 µM forward primer ASFV-F1 (SEQ ID NO: 1), 0.01-2 µM reverse primer ASFV-R1 (SEQ ID NO: 2), 0.01-2 µM probe ASFV-P1 (5' FAM- CACAAGCCGCACCAAAGCAAACCT -BHQ1 3', SEQ ID NO: 3) a commercial RT-PCR kit with a total volume of 20 µl (for example, but not limited to, OneStep PrimeScript ^TM RT-PCR Kit; Takara Bio Inc ., Japan) for real-time PCR analysis. The real-time PCR program is 42 ^o C for 5 minutes, 94 ^o C for 10 seconds, and 40 cycles of 94 ^o C for 10 seconds and 60 ^o C for 30 minutes. Record the results of the fluorescence measurement at 60 ^{o C.}

Real time PCR analysis of the standard curve as shown in FIG. 1, is calculated serially diluted (10 fold) pASFV plastid. 1 copy number of pASFV plastids can be detected. ^{The R 2} value of the standard curve is 0.99812, which indicates that the primer pair and probe of the present invention can be used for real-time PCR and produce credible results.

In addition, another set of primer pairs and probes were used for real-time PCR analysis. To contain 2 µl pASFV plastids (respectively 10 ⁰ , 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ copies), 0.01-2 µM forward primer ASFV-F2 (SEQ ID NO: 4), 0.01-2 µM reverse primer ASFV-R2 (SEQ ID NO: 5), 0.01-2 µM probe ASFV-P2 (5' FAM- TCCTCATCAACACCGAGATTGGCACA -BHQ1 3', SEQ ID NO: 6) A commercial RT-PCR kit (for example, but not limited to, OneStep PrimeScript ^TM RT-PCR Kit; Takara Bio Inc., Japan) with a total volume of 20 µl for real-time PCR analysis. The real-time PCR program is 42 ^o C for 5 minutes, 94 ^o C for 10 seconds, and 40 cycles of 94 ^o C for 10 seconds and 60 ^o C for 30 minutes. Record the results of the fluorescence measurement at 60 ^{o C.}

Real time PCR as shown in Figure 2, is calculated serially diluted (10 fold) pASFV plastid standard curve analysis. At least 10 copies of pASFV plastids can be detected. ^{The R 2} value of the standard curve is 0.9998, which indicates that the primer pair and probe of the present invention can be used for real-time PCR and produce credible results.

The results of Examples 1-3 show that the primer pair and probe of the present invention can be used in different polymerase chain reactions, including traditional PCR, cPCR, qPCR, etc., to detect the presence of African swine fever virus (ASFV), and has a high degree of Sensitivity and specificity.

The above detailed description is a specific description of a possible embodiment of the present invention, but this embodiment is not intended to limit the scope of the patent of the present invention. Any equivalent implementation or modification that does not deviate from the technical spirit of the present invention should be included in In the scope of the patent in this case.

In summary, the technical features disclosed in this case have fully met the statutory requirements for invention patents for novelty and advancement. I filed an application in accordance with the law. I implore your bureau to approve this invention patent application to encourage invention, and it will be convenient.

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

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

Claims (1)

An oligonucleotide pair and probe for detecting African swine fever virus, comprising a first primer, a second primer, and an oligonucleotide probe, the oligonucleotide probe comprising an oligonucleotide Nucleotide sequence, a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence inhibitor molecule attached to a second position on the oligonucleotide probe; the first The sequence combination of a 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; Wherein when the sequence of the first primer and the second primer are combined into SEQ ID NO: 1 and SEQ ID NO: 2, the oligonucleotide sequence is shown in SEQ ID NO: 3; when the first primer and the When the sequence combination of the second primer is SEQ ID NO: 4 and SEQ ID NO: 5, the oligonucleotide sequence is shown in SEQ ID NO: 6; when the sequence of the first primer and the second primer are combined into SEQ ID NO: 6 ID NO: 7 and SEQ ID NO: 5, the oligonucleotide sequence is selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 8; when the sequences of the first primer and the second primer are combined Selected from the group consisting of: 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, and SEQ ID NO: 10 and SEQ ID NO: 13, the oligonucleotide sequence is as SEQ ID NO: 14; and when 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, the oligonucleotide sequence is shown in SEQ ID NO:20.

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