Markers to predict macrocyclic lactone drug resistance in
Disclosed are nucleic acid molecules from the genome of Dirofilaria spp. nematodes that contain single nucleotide polymorphisms related to reduced responsiveness of the nematodes to macrocyclic lactones. In one example, the species of Dirofilaria is Dirofilaria immitis (the agent of heartworm in animals). Also disclosed are methods for determining the responsiveness of Dirofilaria spp. nematodes to macrocyclic lactones, methods for selecting a treatment to treat an animal infected with a Dirofilaria spp. nematode, and kits for determining the responsiveness of Dirofilaria spp. nematodes to macrocyclic lactones.
1. An isolated nucleic acid molecule comprising 25-300 nucleotides of SEQ ID NO: 118, or a reverse complement thereof;
wherein the isolated nucleic acid molecule includes a polymorphic site at position 151 which confers resistance to a macrocyclic lactone, wherein position 151 is G; and
wherein the isolated nucleic acid molecule further includes a detectable label selected from the group consisting of radioactive, enzymatic, and fluorescent labels.
2. The isolated nucleic acid molecule of claim 1 , wherein the nucleic acid molecule has at least 35 nucleotides of SEQ ID NO: 118, or a reverse complement thereof;
wherein the isolated nucleic acid molecule includes a polymorphic site that confers resistance to a macrocyclic lactone.
3. The isolated nucleic acid molecule of claim 1 , wherein the nucleic acid molecule has at least 50 nucleotides of SEQ ID NO: 118, or a reverse complement thereof;
wherein the isolated nucleic acid molecule includes a polymorphic site that confers resistance to a macrocyclic lactone.
4. The isolated nucleic acid molecule of claim 1 , wherein the nucleic acid molecule has at least 100 nucleotides of SEQ ID NO: 118, or a reverse complement thereof;
wherein the isolated nucleic acid molecule includes a polymorphic site that confers resistance to a macrocyclic lactone.
5. The isolated nucleic acid molecule of claim 1 , wherein the nucleic acid molecule has at least 45 nucleotides of SEQ ID NO: 118, or a reverse complement thereof.
6. The isolated nucleic acid molecule of claim 1 , wherein the nucleic acid molecule has at least 30 nucleotides of SEQ ID NO: 118, or a reverse complement thereof.
7. The isolated nucleic acid molecule of claim 1 , wherein the nucleic acid molecule has at least 40 nucleotides of SEQ ID NO: 118, or a reverse complement thereof.
8. A kit for determining a genotype of a Dirofilaria spp. nematode, the kit comprising at least a first isolated nucleic acid molecule comprising 25-300 nucleotides of SEQ ID NO: 118, or a reverse complement thereof;
wherein the isolated nucleic acid molecule includes a polymorphic site at position 151 which confers resistance to a macrocyclic lactone, wherein position 151 is G; and
wherein the oligonucleotide further includes a detectable label selected from the group consisting of radioactive, enzymatic, and fluorescent labels.
9. The kit of claim 8 , wherein the isolated nucleic acid molecule is a probe, a primer or an aptamer.
10. The kit of claim 8 , wherein the kit further comprises one or more of reagents, buffers, packaging materials, and instructions for determining the genotype of the nematode by DNA sequencing, hybridization-based methods including with allele specific oligonucleotides, microarray analysis, enzyme-based methods, single strand conformational polymorphism (SSCP), high resolution melt (HRM) or approaches based on PCR, RT-PCR, or qRT-PCR.
11. The kit of claim 8 , further comprising at least a second isolated nucleic acid molecule having a length of at least 10 nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 1-113, 115-117, and 119-127, or a reverse complement thereof;
wherein the at least a second isolated nucleic acid molecule includes a disclosed polymorphic site that correlates with resistance to a macrocyclic lactone; and
wherein the at least a second isolated nucleic acid molecule further includes a detectable label.