US 2021/0071231 A1 · PRIMER COMBINATION FOR DETECTING 9 NA SUBTYPE AIVS AND APPLICATION THEREOF

Abstract

The present invention discloses a primer combination for detecting 9 NA subtype AIVs and an application thereof. The present invention protects a primer combination comprising 10 primer pairs (20 primers), and the 20 primers are set forth in SEQ ID NO: 1 to SEQ ID NO: 20 of the Sequence Listing in sequence. When used for identifying 9 NA subtypes, a GeXP detection method in combination with the primer combination provided by the present invention has the advantages of high throughput, fast, time-saving and labor-saving. The present invention provides an effective method for rapid identification of 9 different NA subtype AIVs and has a broad application prospect.

A primer combination, which comprises the following 10 primer pairs:

primer pair AIV-M, which consists of a forward primer and a reverse primer, wherein the forward primer is (a1), (a2), (a3) or (a4) and the reverse primer is (b1), (b2), (b3) or (b4);
(a1) a primer with the nucleotide sequence set forth in SEQ ID NO:
1 of the Sequence Listing;
(a2) a primer with the nucleotide sequence set forth in positions 19-38 of SEQ ID NO:
1 of the Sequence Listing;
(a3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(a4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (a3) and/or (a4) have the nucleotide sequence set forth in positions 19-38 of SEQ ID NO:
1 of the Sequence Listing;
(b1) a primer with the nucleotide sequence set forth in SEQ ID NO:
2 of the Sequence Listing;
(b2) a primer with the nucleotide sequence set forth in positions 20-39 of SEQ ID NO:
2 of the Sequence Listing;
(b3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(b4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (b3) and/or (b4) have the nucleotide sequence set forth in positions 20-39 of SEQ ID NO:
2 of the Sequence Listing;
primer pair N1, which consists of a forward primer and a reverse primer, wherein the forward primer is (i1), (i2), (i3) or (i4) and the reverse primer is (1), (j2), (j3) or (j4);
(i1) a primer with the nucleotide sequence set forth in SEQ ID NO:
3 of the Sequence Listing;
(i2) a primer with the nucleotide sequence set forth in positions 19-38 of SEQ ID NO:
93 of the Sequence Listing;
(i3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(i4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (i3) and/or (i4) have the nucleotide sequence set forth in positions 19-38 of SEQ ID NO:
3 of the Sequence Listing;
(1) a primer with the nucleotide sequence set forth in SEQ ID NO:
4 of the Sequence Listing;
(j2) a primer with the nucleotide sequence set forth in positions 20-38 of SEQ ID NO:
4 of the Sequence Listing;
(j3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(j4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (3) and/or (4) have the nucleotide sequence set forth in positions 20-38 of SEQ ID NO:
4 of the Sequence Listing;
primer pair N2, which consists of a forward primer and a reverse primer, wherein the forward primer is (k1), (k2), (k3) or (k4) and the reverse primer is (11), (12), (13) or (14);
(k1) a primer with the nucleotide sequence set forth in SEQ ID NO:
5 of the Sequence Listing;
(k2) a primer with the nucleotide sequence set forth in positions 19-35 of SEQ ID NO:
5 of the Sequence Listing;
(k3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(k4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (k3) and/or (k4) have the nucleotide sequence set forth in positions 19-35 of SEQ ID NO:
5 of the Sequence Listing;
(l1) a primer with the nucleotide sequence set forth in SEQ ID NO:
6 of the Sequence Listing;
(l2) a primer with the nucleotide sequence set forth in positions 20-36 of SEQ ID NO:
6 of the Sequence Listing;
(l3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(l4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (13) and/or (14) have the nucleotide sequence set forth in positions 20-36 of SEQ ID NO:
6 of the Sequence Listing;
primer pair N3, which consists of a forward primer and a reverse primer, wherein the forward primer is (m1), (m2), (m3) or (m4) and the reverse primer is (n1), (n2), (n3) or (n4);
(m1) a primer with the nucleotide sequence set forth in SEQ ID NO:
7 of the Sequence Listing;
(m2) a primer with the nucleotide sequence set forth in positions 19-41 of SEQ ID NO:
7 of the Sequence Listing;
(m3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(m4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (m3) and/or (m4) have the nucleotide sequence set forth in positions 19-41 of SEQ ID NO:
7 of the Sequence Listing;
(n1) a primer with the nucleotide sequence set forth in SEQ ID NO:
8 of the Sequence Listing;
(n2) a primer with the nucleotide sequence set forth in positions 20-41 of SEQ ID NO:
8 of the Sequence Listing;
(n3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(n4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (n3) and/or (n4) have the nucleotide sequence set forth in positions 20-41 of SEQ ID NO:
8 of the Sequence Listing;
primer pair N4, which consists of a forward primer and a reverse primer, wherein the forward primer is (01), (o2), (o3) or (o4) and the reverse primer is (p1), (p2), (p3) or (p4);
(o1) a primer with the nucleotide sequence set forth in SEQ ID NO:
9 of the Sequence Listing;
(o2) a primer with the nucleotide sequence set forth in positions 19-40 of SEQ ID NO:
9 of the Sequence Listing;
(o3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(o4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (o3) and/or (o4) have the nucleotide sequence set forth in positions 19-40 of SEQ ID NO:
9 of the Sequence Listing;
(p1) a primer with the nucleotide sequence set forth in SEQ ID NO:
10 of the Sequence Listing;
(p2) a primer with the nucleotide sequence set forth in positions 20-39 of SEQ ID NO:
10 of the Sequence Listing;
(p3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(p4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (p3) and/or (p4) have the nucleotide sequence set forth in positions 20-39 of SEQ ID NO:
10 of the Sequence Listing;
primer pair N5, which consists of a forward primer and a reverse primer, wherein the forward primer is (q1), (q2), (q3) or (q4) and the reverse primer is (r1), (r2), (r3) or (r4);
(q1) a primer with the nucleotide sequence set forth in SEQ ID NO:
11 of the Sequence Listing;
(q2) a primer with the nucleotide sequence set forth in positions 19-38 of SEQ ID NO:
11 of the Sequence Listing;
(q3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(q4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (q3) and/or (q4) have the nucleotide sequence set forth in positions 19-38 of SEQ ID NO:
11 of the Sequence Listing;
(r1) a primer with the nucleotide sequence set forth in SEQ ID NO:
12 of the Sequence Listing;
(r2) a primer with the nucleotide sequence set forth in positions 20-40 of SEQ ID NO:
12 of the Sequence Listing;
(r3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(r4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (r3) and/or (r4) have the nucleotide sequence set forth in positions 20-40 of SEQ ID NO:
12 of the Sequence Listing;
primer pair N6, which consists of a forward primer and a reverse primer, wherein the forward primer is (s1), (s2), (s3) or (s4) and the reverse primer is (t1), (t2), (t3) or (t4);
(s1) a primer with the nucleotide sequence set forth in SEQ ID NO:
13 of the Sequence Listing;
(s2) a primer with the nucleotide sequence set forth in positions 19-43 of SEQ ID NO:
13 of the Sequence Listing;
(s3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(s4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (s3) and/or (s4) have the nucleotide sequence set forth in positions 19-43 of SEQ ID NO:
13 of the Sequence Listing;
(t1) a primer with the nucleotide sequence set forth in SEQ ID NO:
14 of the Sequence Listing;
(t2) a primer with the nucleotide sequence set forth in positions 20-44 of SEQ ID NO:
14 of the Sequence Listing;
(t3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(t4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (t3) and/or (t4) have the nucleotide sequence set forth in positions 20-44 of SEQ ID NO:
14 of the Sequence Listing;
primer pair N7, which consists of a forward primer and a reverse primer, wherein the forward primer is (u1), (u2), (u3) or (u4) and the reverse primer is (v1), (v2), (v3) or (v4);
(u1) a primer with the nucleotide sequence set forth in SEQ ID NO:
15 of the Sequence Listing;
(u2) a primer with the nucleotide sequence set forth in positions 19-38 of SEQ ID NO:
15 of the Sequence Listing;
(u3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(u4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (u3) and/or (u4) have the nucleotide sequence set forth in positions 19-38 of SEQ ID NO:
15 of the Sequence Listing;
(v1) a primer with the nucleotide sequence set forth in SEQ ID NO:
16 of the Sequence Listing;
(v2) a primer with the nucleotide sequence set forth in positions 20-39 of SEQ ID NO:
16 of the Sequence Listing;
(v3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(v4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (v3) and/or (v4) have the nucleotide sequence set forth in positions 20-39 of SEQ ID NO:
16 of the Sequence Listing;
primer pair N8, which consists of a forward primer and a reverse primer, wherein the forward primer is (w1), (w2), (w3) or (w4) and the reverse primer is (x1), (x2), (x3) or (x4);
(w1) a primer with the nucleotide sequence set forth in SEQ ID NO:
17 of the Sequence Listing;
(w2) a primer with the nucleotide sequence set forth in positions 19-38 of SEQ ID NO:
17 of the Sequence Listing;
(w3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(w4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (w3) and/or (w4) have the nucleotide sequence set forth in positions 19-38 of SEQ ID NO:
17 of the Sequence Listing;
(x1) a primer with the nucleotide sequence set forth in SEQ ID NO:
18 of the Sequence Listing;
(x2) a primer with the nucleotide sequence set forth in positions 20-40 of SEQ ID NO:
18 of the Sequence Listing;
(x3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(x4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (x3) and/or (x4) have the nucleotide sequence set forth in positions 20-40 of SEQ ID NO:
18 of the Sequence Listing;
primer pair N9, which consists of a forward primer and a reverse primer, wherein the forward primer is (y1), (y2), (y3) or (y4) and the reverse primer is (z1), (z2), (z3) or (z4);
(y1) a primer with the nucleotide sequence set forth in SEQ ID NO:
19 of the Sequence Listing;
(y2) a primer with the nucleotide sequence set forth in positions 19-39 of SEQ ID NO:
19 of the Sequence Listing;
(y3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(y4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (y3) and/or (y4) have the nucleotide sequence set forth in positions 19-39 of SEQ ID NO:
19 of the Sequence Listing;
(z1) a primer with the nucleotide sequence set forth in SEQ ID NO:
20 of the Sequence Listing;
(z2) a primer with the nucleotide sequence set forth in positions 20-39 of SEQ ID NO:
20 of the Sequence Listing;
(z3) a primer comprising the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
(z4) a primer consisting of the following segments in order from upstream to downstream:
a GeXP universal primer and a gene-specific primer;
wherein, the gene-specific primers in (z3) and/or (z4) have the nucleotide sequence set forth in positions 20-39 of SEQ ID NO:
20 of the Sequence Listing.

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Primer combination for detecting 9 na subtype aivs and application thereof

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Primer combination for detecting 9 na subtype aivs and application thereof

Life of this patent

Abstract

Claims5