TWI731425B - Pairs of oligonucleotides and probes for detecting african swine fever virus (asfv) - Google Patents
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Abstract
Description
本發明關於檢測非洲豬瘟病毒(African Swine Fever Virus,ASFV)的方法,特別是關於使用寡核苷酸對檢測非洲豬瘟病毒(ASFV)的方法。 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.
豬是全球最重要的經濟動物之一。根據美國農業部統計,2018年全球主要養豬國的豬肉總產量達113,081千噸。豬肉的經濟價值已超過千億美金。然而,密集養殖加上管理不良可能造成豬隻容易感染疾病且疫情迅速擴散。以2018年中國爆發的非洲豬瘟(African Swine Fever,ASF)為例,病豬死亡率幾乎為100%,已經造成了巨大的經濟損失。由此可見,在飼養場密集監控豬隻疾病對管理而言是不可或缺的一環。本發明即提供了快速、方便的非洲豬瘟病毒(ASFV)檢測方法以供產業利用。 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.
於一方面,本發明涉及一種非洲豬瘟病毒(ASFV)檢測方法,包含提供一可能含有一非洲豬瘟病毒(ASFV)的一或多個核苷酸序列的樣本;提供一寡核苷酸引子對,該寡核苷酸引子對包含一第一引子與一第二引子,該寡核列的二互補股的5’端;提供一聚合酶;在一容器中混合該樣本、該寡核苷酸引子對、該聚合酶、去氧腺苷三磷酸(deoxyadenosine triphosphates, dATPs)、去氧胞核苷三磷酸(deoxycytidine triphosphates, dCTPs)、去氧鳥苷三磷酸(deoxyguanosine triphosphates, dGTPs),以及去氧胸苷三磷酸(deoxythymidine triphosphates, dTTPs),以形成一聚合酶連鎖反應(polymerase chain reaction, PCR)混合物;透過在一固定溫度下加熱該容器的底部,使該PCR混合物進行熱對流聚合酶連鎖反應(convective polymerase chain reaction, cPCR),以形成一PCR產物;以及偵測該PCR產物以辨識該雙股目標序列。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.
在某些具體實施例中,該第一引子與該第二引子的序列組合選自包含以下之群組:SEQ ID NO: 1與SEQ ID NO: 2、SEQ ID NO: 4與SEQ ID NO: 5、SEQ ID NO: 7與SEQ ID NO: 5、SEQ ID NO: 9與SEQ ID NO: 11、SEQ ID NO: 9與SEQ ID NO: 12、SEQ ID NO: 9與SEQ ID NO: 13、SEQ ID NO: 10與SEQ ID NO: 11、SEQ ID NO: 10與SEQ ID NO: 12、SEQ ID NO: 10與SEQ ID NO: 13、SEQ ID NO: 15與SEQ ID NO: 18、SEQ ID NO: 15與SEQ ID NO: 19、SEQ ID NO: 16與SEQ ID NO: 18、SEQ ID NO: 16與SEQ ID NO: 19、SEQ ID NO: 17與SEQ ID NO: 18,以及SEQ ID NO: 17與SEQ ID NO: 19。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.
在某些具體實施例中,該聚合酶連鎖反應(PCR)混合物進一步包含一寡核苷酸探針,該寡核苷酸探針包含一與該雙股目標序列的一區段互補的序列、一附加於該寡核苷酸探針上的一第一位置的螢光分子,以及一附加於該寡核苷酸探針上的一第二位置的螢光抑制分子,當該寡核苷酸探針未雜合於該雙股目標序列的該區段時,該螢光抑制分子大體上抑制該螢光分子,且當該寡核苷酸探針雜合於該雙股目標序列的該區段時,該螢光分子大體上未被抑制。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.
在某些具體實施例中,該第一引子的序列如SEQ ID NO: 1所示,該第二引子的序列如SEQ ID NO: 2所示。在某些具體實施例中,該聚合酶連鎖反應(PCR)混合物進一步包含一寡核苷酸探針,其序列為一介於該非洲豬瘟病毒(ASFV)的p72完整基因序列 (GenBank accession No. MH713612)的第1162至第1206個核苷酸之間的13至30個鹼基對的寡核苷酸序列。在某些較佳具體實施例中,該寡核苷酸探針的序列如SEQ ID NO: 3所示。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.
在某些具體實施例中,該第一引子的序列如SEQ ID NO: 4所示,該第二引子的序列如SEQ ID NO: 5所示。在某些具體實施例中,該聚合酶連鎖反應(PCR)混合物進一步包含一寡核苷酸探針,其序列為一介於該非洲豬瘟病毒(ASFV)的p72完整基因序列 (GenBank accession No. MH713612)的第454至第503個核苷酸之間的13至30個鹼基對的寡核苷酸序列。在某些較佳具體實施例中,該寡核苷酸探針的序列如SEQ ID NO: 6所示。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.
在某些具體實施例中,該第一引子的序列如SEQ ID NO: 7所示,該第二引子的序列如SEQ ID NO: 5所示。在某些具體實施例中,該聚合酶連鎖反應(PCR)混合物進一步包含一寡核苷酸探針,其序列為一介於該非洲豬瘟病毒(ASFV)的p72完整基因序列 (GenBank accession No. MH713612)的第454至第583個核苷酸之間的13至30個鹼基對的寡核苷酸序列。在某些較佳具體實施例中,該寡核苷酸探針的序列選自由下列所組成之群組:SEQ ID NO: 6以及SEQ ID NO: 8。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.
在某些具體實施例中,該第一引子與該第二引子的序列組合選自包含以下之群組:SEQ ID NO: 9與SEQ ID NO: 11、SEQ ID NO: 9與SEQ ID NO: 13、SEQ ID NO: 10與SEQ ID NO: 11,以及SEQ ID NO: 10與SEQ ID NO: 13。在某些具體實施例中,該聚合酶連鎖反應(PCR)混合物進一步包含一寡核苷酸探針,其序列為一介於該非洲豬瘟病毒(ASFV)的p72完整基因序列 (GenBank accession No. MH713612)的第403至第442個核苷酸之間的13至30個鹼基對的寡核苷酸序列。在某些較佳具體實施例中,該寡核苷酸探針的序列如SEQ ID NO: 14所示。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.
在某些具體實施例中,該第一引子的序列選自包含以下之群組:SEQ ID NO: 9以及SEQ ID NO: 10,該第二引子的序列如SEQ ID NO: 12所示。在某些具體實施例中,該聚合酶連鎖反應(PCR)混合物進一步包含一寡核苷酸探針,其序列為一介於該非洲豬瘟病毒(ASFV)的p72完整基因序列 (GenBank accession No. MH713612)的第403至第453個核苷酸之間的13至30個鹼基對的寡核苷酸序列。在某些較佳具體實施例中,該寡核苷酸探針的序列如SEQ ID NO: 14所示。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.
在某些具體實施例中,該第一引子與該第二引子的序列組合選自包含以下之群組:SEQ ID NO: 15與SEQ ID NO: 18、SEQ ID NO: 15與SEQ ID NO: 19、SEQ ID NO: 16與SEQ ID NO: 18、SEQ ID NO: 16與SEQ ID NO: 19、SEQ ID NO: 17與SEQ ID NO: 18,以及SEQ ID NO: 17與SEQ ID NO: 19。在某些具體實施例中,該聚合酶連鎖反應(PCR)混合物進一步包含一寡核苷酸探針,其序列為一介於該非洲豬瘟病毒(ASFV)的p72完整基因序列 (GenBank accession No. MH713612)的第156至第189個核苷酸之間的13至30個鹼基對的寡核苷酸序列。在某些較佳具體實施例中,該寡核苷酸探針的序列如SEQ ID NO: 20所示。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.
於一方面,本發明涉及一種用於偵測非洲豬瘟病毒(ASFV)的寡核苷酸對,包含一第一引子與一第二引子。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.
在某些具體實施例中,該第一引子的序列如SEQ ID NO: 1所示,該第二引子的序列如SEQ ID NO: 2所示。在某些具體實施例中,該寡核苷酸對進一步包含一寡核苷酸探針,該寡核苷酸探針包含一介於該非洲豬瘟病毒(ASFV)的p72完整基因序列 (GenBank accession No. MH713612)的第1162至第1206個核苷酸之間的13至30個鹼基對的寡核苷酸序列、一附加於該寡核苷酸探針上的一第一位置的螢光分子,以及一附加於該寡核苷酸探針上的一第二位置的螢光抑制分子。在某些較佳具體實施例中,該寡核苷酸探針的序列如SEQ ID NO: 3所示。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.
在某些具體實施例中,該第一引子的序列如SEQ ID NO: 4所示,該第二引子的序列如SEQ ID NO: 5所示。在某些具體實施例中,該寡核苷酸對進一步包含一寡核苷酸探針,該寡核苷酸探針包含一介於該非洲豬瘟病毒(ASFV)的p72完整基因序列 (GenBank accession No. MH713612)的第454至第503個核苷酸之間的13至30個鹼基對的寡核苷酸序列、一附加於該寡核苷酸探針上的一第一位置的螢光分子,以及一附加於該寡核苷酸探針上的一第二位置的螢光抑制分子。在某些較佳具體實施例中,該寡核苷酸探針的序列如SEQ ID NO: 6所示。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.
在某些具體實施例中,該第一引子的序列如SEQ ID NO: 7所示,該第二引子的序列如SEQ ID NO: 5所示。在某些具體實施例中,該寡核苷酸對進一步包含一寡核苷酸探針,該寡核苷酸探針包含一介於該非洲豬瘟病毒(ASFV)的p72完整基因序列 (GenBank accession No. MH713612)的第454至第583個核苷酸之間的13至30個鹼基對的寡核苷酸序列、一附加於該寡核苷酸探針上的一第一位置的螢光分子,以及一附加於該寡核苷酸探針上的一第二位置的螢光抑制分子。在某些較佳具體實施例中,該寡核苷酸探針的序列選自由下列所組成之群組:SEQ ID NO: 6以及SEQ ID NO: 8。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.
在某些具體實施例中,該第一引子與該第二引子的序列組合選自包含以下之群組:SEQ ID NO: 9與SEQ ID NO: 11、SEQ ID NO: 9與SEQ ID NO: 13、SEQ ID NO: 10與SEQ ID NO: 11,以及SEQ ID NO: 10與SEQ ID NO: 13。在某些具體實施例中,該寡核苷酸對進一步包含一寡核苷酸探針,該寡核苷酸探針包含一介於該非洲豬瘟病毒(ASFV)的p72完整基因序列 (GenBank accession No. MH713612)的第403至第442個核苷酸之間的13至30個鹼基對的寡核苷酸序列、一附加於該寡核苷酸探針上的一第一位置的螢光分子,以及一附加於該寡核苷酸探針上的一第二位置的螢光抑制分子。在某些較佳具體實施例中,該寡核苷酸探針的序列如SEQ ID NO: 14所示。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.
在某些具體實施例中,該第一引子的序列選自包含以下之群組:SEQ ID NO: 9以及SEQ ID NO: 10,該第二引子的序列如SEQ ID NO: 12所示。在某些具體實施例中,該寡核苷酸對進一步包含一寡核苷酸探針,該寡核苷酸探針包含一介於該非洲豬瘟病毒(ASFV)的p72完整基因序列 (GenBank accession No. MH713612)的第403至第453個核苷酸之間的13至30個鹼基對的寡核苷酸序列、一附加於該寡核苷酸探針上的一第一位置的螢光分子,以及一附加於該寡核苷酸探針上的一第二位置的螢光抑制分子。在某些較佳具體實施例中,該寡核苷酸探針的序列如SEQ ID NO: 14所示。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.
在某些具體實施例中,該第一引子與該第二引子的序列組合選自包含以下之群組:SEQ ID NO: 15與SEQ ID NO: 18、SEQ ID NO: 15與SEQ ID NO: 19、SEQ ID NO: 16與SEQ ID NO: 18、SEQ ID NO: 16與SEQ ID NO: 19、SEQ ID NO: 17與SEQ ID NO: 18,以及SEQ ID NO: 17與SEQ ID NO: 19。在某些具體實施例中,該寡核苷酸對進一步包含一寡核苷酸探針,該寡核苷酸探針包含一介於該非洲豬瘟病毒(ASFV)的p72完整基因序列 (GenBank accession No. MH713612)的第156至第189個核苷酸之間的13至30個鹼基對的寡核苷酸序列、一附加於該寡核苷酸探針上的一第一位置的螢光分子,以及一附加於該寡核苷酸探針上的一第二位置的螢光抑制分子。在某些較佳具體實施例中,該寡核苷酸探針的序列如SEQ ID NO: 20所示。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.
於一方面,本發明涉及一種非洲豬瘟病毒(ASFV)檢測方法。在某些具體實施例中,該方法為聚合酶連鎖反應(polymerase chain reaction, PCR)。在某些具體實施例中,該方法為反轉錄聚合酶連鎖反應(reverse-transcription polymerase chain reaction, RT-PCR)。在某些具體實施例中,該方法為熱對流聚合酶連鎖反應(convective polymerase chain reaction, cPCR)。在某些具體實施例中,該方法為即時聚合酶連鎖反應(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). 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).
於一方面,本發明涉及一種非洲豬瘟病毒(ASFV)檢測方法,包含: 提供一可能含有一非洲豬瘟病毒(ASFV)的一或多個核苷酸序列的樣本; 提供一寡核苷酸引子對,該寡核苷酸引子對包含一第一引子與一第二引子,該寡核苷酸引子對定義在該非洲豬瘟病毒(ASFV)的一或多個核苷酸序列上一雙股目標序列的二互補股的5’端; 提供一聚合酶; 在一容器中混合該樣本、該寡核苷酸引子對、該聚合酶、去氧腺苷三磷酸(dATPs)、去氧胞核苷三磷酸(dCTPs)、去氧鳥苷三磷酸(dGTPs),以及去氧胸苷三磷酸(dTTPs),以形成一聚合酶連鎖反應(PCR)混合物; 透過在一固定溫度下加熱該容器的底部,使該PCR混合物進行熱對流聚合酶連鎖反應(cPCR),以形成一PCR產物;以及 偵測該PCR產物以辨識該雙股目標序列。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.
於另一方面,本發明涉及一種非洲豬瘟病毒(ASFV)檢測方法,包含: 提供一可能含有一非洲豬瘟病毒(ASFV)的一或多個核苷酸序列的樣本; 提供一寡核苷酸引子對,該寡核苷酸引子對包含一第一引子與一第二引子,該寡核苷酸引子對定義在該非洲豬瘟病毒(ASFV)的一或多個核苷酸序列上一雙股目標序列的二互補股的5’端; 提供一寡核苷酸探針,該寡核苷酸探針包含一與該雙股目標序列的一區段互補的序列、一附加於該寡核苷酸探針上的一第一位置的螢光分子,以及一附加於該寡核苷酸探針上的一第二位置的螢光抑制分子,當該寡核苷酸探針未雜合於該雙股目標序列的該區段時,該螢光抑制分子大體上抑制該螢光分子,且當該寡核苷酸探針雜合於該雙股目標序列的該區段時,該螢光分子大體上未被抑制; 提供一聚合酶; 在一容器中混合該樣本、該寡核苷酸引子對、該寡核苷酸探針、該聚合酶、去氧腺苷三磷酸(dATPs)、去氧胞核苷三磷酸(dCTPs)、去氧鳥苷三磷酸(dGTPs),以及去氧胸苷三磷酸(dTTPs),以形成一聚合酶連鎖反應(PCR)混合物; 透過在一固定溫度下加熱該容器的底部,使該PCR混合物進行熱對流聚合酶連鎖反應(cPCR),以形成一PCR產物;以及 偵測該PCR產物以辨識該雙股目標序列。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.
於又一方面,本發明涉及一種用於偵測非洲豬瘟病毒(ASFV)的寡核苷酸對。In yet another aspect, the present invention relates to an oligonucleotide pair for detecting African swine fever virus (ASFV).
於再一方面,本發明涉及一種用於偵測非洲豬瘟病毒(ASFV)的寡核苷酸對與寡核苷酸探針。In another aspect, the present invention relates to an oligonucleotide pair and an oligonucleotide probe for detecting African swine fever virus (ASFV).
應當進一步理解的是,在某些具體實施例中,本文揭露的寡核苷酸對及/或寡核苷酸探針可用於各種基礎PCR技術之變異,例如,但不限於,反轉錄聚合酶連鎖反應(RT-PCR)、熱對流聚合酶連鎖反應(cPCR)、即時聚合酶連鎖反應(real-time PCR)、巢式聚合酶連鎖反應(nested PCR),以及熱不對稱性交錯聚合酶連鎖反應(thermal asymmetric interlaced PCR, TAIL-PCR)。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).
如本文所用,術語「熱對流聚合酶連鎖反應(cPCR)」是指一種聚合酶連鎖反應,其中,將一裝有一PCR樣品的管狀容器底部嵌入一穩定熱源中,並控制該PCR的參數,包括該PCR樣品的總體積、黏度、表面溫度,以及該管狀容器的內徑,使得該PCR樣品的底部到頂部的溫度梯度下降,誘導熱對流並且使得PCR樣品的變性、黏合、聚合在該管狀容器的不同區域中依序且重複發生。熱對流聚合酶連鎖反應(cPCR)的詳細描述請參見如美國專利號8,187,813,其以引用的方式將其整體併入本文。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.
如本文所用,術語「螢光分子」意指一物質或其一部份,其係能夠在可偵測的範圍內顯示螢光。如本文所用,術語「螢光抑制分子」意指一物質或其一部份,其係能夠抑制當由一光源激發時由該螢光分子所發射的螢光。在某些具體實施例中,術語「螢光分子」與「螢光抑制分子」為TaqMan™分析套組(Applied Biosystems Inc.,加州,美國)的螢光分子與螢光抑制分子。TaqMan™分析套組的詳細描述請參見如,Holland et al., Proc. Natl. Acad. Sci, U.S.A. (1991) 88:7276- 7280;美國專利號5,538,848、5,723,591、5,876,930,以及7,413,708皆以引用的方式將其整體併入本文。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.
該螢光分子的例子包括,但不限於,3-(ε-羧)-3'-乙基-5,5'-二甲基己羰花青(3-(ε-carboxypentyl)-3'-ethyl-5,5'-dimethyloxa-carbocyanine, CYA)、6-羧基螢光素(6-carboxyfluorescein, FAM)、5,6-羧基羅丹明-L LO (5,6-carboxyrhodamine-l lO, R110)、6羧基羅丹明-6G (6- carboxyrhodamine-6G, R6G)、N’,N’,N’,N’
-四甲基-6-羧基羅丹明(N’,N’,N’,N’
-tetramethyl-6-carboxyrhodamine, TAMRA)、6-羧基-X-羅丹明(6-carboxy-X-rhodamine, ROX)、2’,4’,5’,7’-四氯4-7-二氯螢光素(2’,4’,5’,7’-tetrachloro-4-7-dichlorofluorescein, TET)、2',7-二甲氧基-4',5'-6羧基羅丹明(2’,7-dimethoxy-4’,5’-6 carboxyrhodamine, JOE)、6-羧基-2',4,4',5',7,7'-六氯熒光素(6-carboxy-2’,4,4’,5’,7,7’-hexachlorofluorescein, HEX)、ALEXA螢光、Cy3螢光與Cy5螢光。該螢光抑制分子的例子包括,但不限於,4-(4'-二甲基氨基-苯偶氮基)苯甲酸(4-(4’-dimethylamino-phenylazo)-benzoic acid, Dabcyl)、黑洞螢光抑制劑1 (Black Hole Quencher 1, BHQ1)、黑洞螢光抑制劑2 (Black Hole Quencher 2, BHQ2)、黑洞螢光抑制劑3 (Black Hole Quencher 3, BHQ3)、二氫環吡咯並吲哚三肽小溝結合物(dihydro cyclo pyrrolo indole tripeptide minor groove binder, MGB)、四甲基羅丹明(tetramethylrhodamine, TAMRA)。在某些具體實施例中,該螢光分子為6-羧基螢光素(FAM),且該螢光抑制分子為二氫環吡咯並吲哚三肽小溝結合物(MGB)。在某些具體實施例中,該螢光分子為6-羧基螢光素(FAM),且該螢光抑制分子為黑洞螢光抑制劑1 (BHQ1)。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 (
除非本文另有定義,否則用以與本文結合的科學與技術術語應具有本領域普通技術人員通常理解的含義。此外,除非上下文另有要求,單數術語應包括複數,並且複數術語應包括單數。本發明的方法與技術一般可根據本領域已知的常規方法進行。一般而言,本文所描述之用以連結以下技術的命名法,以及生物化學、酵素學、分子及細胞生物學、微生物學、遺傳學與蛋白質及核酸化學及雜合反應的技術皆為本領域已知且經常使用者。除非另有說明,本發明的方法與技術一般可根據本領域已知的常規方法進行,且被描述於在本說明書中被引用且討論的各種一般及更具體的參考文獻中。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.
本發明進一步透過以下的實施例闡釋,其不應以任何方式被解釋為進一步的限縮。本申請案中引用的所有引用文件(包括參考文獻、核准的專利、公開的專利申請,以及一同在申請中的專利申請案)的整體內容,在此透過引用的方式明確地併入本案中。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.
實施例Example
實施例Example 11 以傳統聚合酶連鎖反應檢測非洲豬瘟病毒Detection of African swine fever virus with traditional polymerase chain reaction (ASFV)(ASFV)
非洲豬瘟病毒(ASFV)的p72基因(GenBank accession No. MH713612)全長序列被插入一選殖載體中,該選殖載體可為,但不限於,pUC57、pGEM-T,以得到pASFV質體。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.
進行傳統PCR所用的50 µl PCR混合物含有:106
個拷貝數的pASFV質體、0.01-2 µM正向引子、0.01-2 µM反向引子、0.2 µM dNTP以及1.25U Taq DNA聚合酶。在一熱循環儀(例如,但不限於PC818, Astec Co. Ltd.,日本)中進行擴增反應,且包含一個變性的初始循環94o
C持續3分鐘,以及35個循環的94o
C 30秒、60o
C 30秒以及72o
C延展30秒。擴增的產物接著以15%聚丙烯醯胺凝膠(polyacrylamide gel)在TAE緩衝液(40 mM Tris, 20 mM acetic acid, 1 mM EDTA)中分析,並且以溴化乙錠(ethidium bromide)染色顯現。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
傳統PCR的結果如表1所示,各引子對擴增了各目標序列的正確大小的片段,而在負對照組中則無目標序列被擴增(結果未顯示)。該結果表明,各引子對可用於傳統PCR擴增反應以檢測非洲豬瘟病毒(ASFV)的存在。
【表1】各引子對進行傳統PCR的結果
實施例Example 22 以熱對流聚合酶連鎖反應Thermal convective polymerase chain reaction (cPCR)(cPCR) 檢測非洲豬瘟病毒Detection of African swine fever virus (ASFV)(ASFV)
進行熱對流聚合酶連鎖反應(cPCR)所用的50 µl PCR混合物含有:帶有非洲豬瘟病毒(ASFV)的p72基因全長序列的質體(pASFV質體,同實施例1)(分別為102
, 103
, 104
, 105
, 106
拷貝數)、0.01-2 µM正向引子、0.01-2 µM反向引子、0.01-2 µM探針(各探針序列的5’端接合一螢光分子6-羧基螢光素(FAM),且各探針序列的3’端接合一螢光抑制分子黑洞螢光抑制劑1 (BHQ1)或四甲基羅丹明(TAMRA))、0.2 µM dNTP、1X cPCR緩衝液,以及1-5 U Taq DNA聚合酶。將PCR混合物加入一反應試管中,並置於一熱對流聚合酶連鎖反應(cPCR)儀中一段指定的時間(約30~45分鐘)。以該cPCR儀偵測每個樣本中的FAM螢光。重複上述cPCR分析試驗8次(n = 8)以評各估引子對與探針的敏感度。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
敏感度測試的結果如表2所示,各引子對及探針的組合皆可100%正確偵測到樣品中含有102
拷貝數的pASFV質體,其敏感度可達102
拷貝數。
【表2】各引子對與探針組合的敏感度測試結果(n = 8)
此外,以不同的豬病原菌基因質體(106
拷貝數/µl)作為cPCR模板分析上述各引子對及探針組合的專一性。cPCR方法如上所述。專一性測試的結果如表3所示,各引子對及探針的組合皆可正確偵測到含有非洲豬瘟病毒(ASFV)的樣品,而偵測不到含有典型豬瘟病毒(classical swine fever virus, CSFV)、口蹄疫病毒(Foot and mouth disease virus, FMDV)、日本腦炎病毒(Japanese Encephalitis Virus, JEV)、豬繁殖與呼吸道症候群病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)、A型豬流感病毒(Swine influenza A virus, SIV)、豬流行性下痢病毒(Porcine epidemic diarrhea virus, PEDV)、豬環狀病毒第二型(Porcine circovirus type 2, PCV2)、偽狂犬病病毒(Pseudorabies virus, PRV)的樣品。該結果顯示各引子對及探針組合具有專一性。
【表3】各引子對與探針組合的專一性測試結果
實施例Example 33 以即時聚合酶連鎖反應Real-time polymerase chain reaction (real-time PCR, qPCR)(real-time PCR, qPCR) 檢測非洲豬瘟病毒Detection of African swine fever virus (ASFV)(ASFV)
以稀釋的pASFV質體(分別為100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 個拷貝數)於一即時PCR儀(例如,但不限於ABI StepOnePlusTM ; Applied BioSystem, Life Technologies,加州,美國)中進行即時PCR分析。以含有2 µl pASFV質體、0.01-2 µM正向引子ASFV-F1 (SEQ ID NO: 1)、0.01-2 µM反向引子ASFV-R1 (SEQ ID NO: 2)、0.01-2 µM探針ASFV-P1 (5’ FAM- CACAAGCCGCACCAAAGCAAACCT -BHQ1 3’, SEQ ID NO: 3)總體積為20 µl的商用RT-PCR套組(例如,但不限於,OneStep PrimeScriptTM RT-PCR Kit; Takara Bio Inc.,日本)進行即時PCR分析。即時PCR的程序為42o C 5分鐘、94o C 10秒,以及40個循環的94o C 10秒以及60o C 30分鐘。在60o C的步驟中記錄螢光測量的結果。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.
如圖 1 所示,計算連續稀釋(10倍)的pASFV質體的即時PCR分析的標準曲線。1個拷貝數的pASFV質體可被偵測到。該標準曲線的R2 值為0.99812,表示本發明的引子對與探針可以被用於即時PCR,並產生可信的結果。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.
此外,以另一組引子對及探針進行即時PCR分析。以含有2 µl pASFV質體(分別為100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 個拷貝數)、0.01-2 µM正向引子ASFV-F2 (SEQ ID NO: 4)、0.01-2 µM反向引子ASFV-R2 (SEQ ID NO: 5)、0.01-2 µM探針ASFV-P2 (5’ FAM- TCCTCATCAACACCGAGATTGGCACA -BHQ1 3’, SEQ ID NO: 6)總體積為20 µl的商用RT-PCR套組(例如,但不限於,OneStep PrimeScriptTM RT-PCR Kit; Takara Bio Inc.,日本)進行即時PCR分析。即時PCR的程序為42o C 5分鐘、94o C 10秒,以及40個循環的94o C 10秒以及60o C 30分鐘。在60o C的步驟中記錄螢光測量的結果。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.
如圖 2 所示,計算連續稀釋(10倍)的pASFV質體的即時PCR分析的標準曲線。至少10個拷貝數的pASFV質體可被偵測到。該標準曲線的R2 值為0.9998,表示本發明的引子對與探針可以被用於即時PCR,並產生可信的結果。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.
由實施例1-3結果表明,本發明的引子對與探針可用於不同的聚合酶連鎖反應,包括傳統PCR、cPCR、qPCR等,以檢測非洲豬瘟病毒(ASFV)的存在,並且具有高度敏感度及專一性。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.
上列詳細說明係針對本發明之一可行實施例之具體說明,惟該實施例並非用以限制本發明之專利範圍,凡未脫離本發明技藝精神所為之等效實施或變更,均應包含於本案之專利範圍中。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.
綜上所述,本案所揭露之技術特徵已充分符合新穎性及進步性之法定發明專利要件,爰依法提出申請,懇請 貴局核准本件發明專利申請案,以勵發明,至感德便。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.
圖 1 所示為使用引子ASFV-F1與ASFV-R1以及探針ASFV-P1進行即時PCR (real-time PCR)檢測非洲豬瘟病毒(ASFV)的結果。 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.
圖 2 所示為使用引子ASFV-F2與ASFV-R2以及探針ASFV-P2進行即時PCR (real-time PCR)檢測非洲豬瘟病毒(ASFV)的結果。 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.
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