Compositions and methods for detecting huanglongbing
The present invention features compositions and methods for detecting Huanglongbing (HLB) in citrus trees and insects. In one aspect, the invention provides a method of detecting a Huanglongbing (HLB) infection in a citrus grove involving obtaining an extract from a biological sample derived from a citrus grove, contacting the extract with forward and reverse primers that specifically bind a Candidutus nucleic acid molecule in the presence of a nicking enzyme, dNTPs, and a polymerase under conditions permissive for the isothermal amplification of the nucleic acid molecule; and detecting a Candidutus amplicon in the extract.
1. A polynucleotide selected from the group consisting of a forward primer comprising
(SEQ ID NO: 1)
5′ GGACTCCATATGGAGTCCTCGCGAGCGG-MeOC-MeOA-MeOG-
MeOA-MeOC 3′,
a reverse primer comprising the nucleic acid sequence
(SEQ ID NO: 2)
5′ TGACTCCATATGGAGTCATCTAGATTCC-MeOU-MeOA-MeOC-
MeOG-MeOC 3′;
and
a probe comprising
(SEQ ID NO: 3)
5′ CACGCA-MeOG-C-MeOA-G-MeOA-C-MeOG-G-MeOG-MeOU-
G-MeOA-G-MeOU-A-MeOA-C-MeOG-TGCGTG 3′.
2. A kit comprising one or more polynucleotides of claim 1 .
3. A method of detecting a Huanglongbing (HLB) infection in a biological sample, the method comprising
obtaining an extract from a biological sample,
contacting the extract with forward and reverse primers that specifically bind a Candidutus nucleic acid molecule in the presence of a nicking enzyme, dNTPs, and a polymerase under conditions permissive for the isothermal amplification of the nucleic acid molecule;
wherein the forward and reverse primers comprise the following sequences, respectively:
(SEQ ID NO: 1)
5′ GGACTCCATATGGAGTCCTCGCGAGCGG-MeOC-MeOA-MeOG-
MeOA-MeOC 3′
and
(SEQ ID NO: 2)
5′ TGACTCCATATGGAGTCATCTAGATTCC-MeOU-MeOA-MeOC-
MeOG-MeOC 3′
and
deleting a Candidutus amplicon in the extract,
wherein the presence of the Candidutus amplicon detects an HLB infection in the sample and failure to detect the amplicon indicated the absence of an HLB infection in the sample.
4. The method of claim 3 , wherein the biological sample comprises plant, insect, or plant and insect material.
5. The method of claim 4 , wherein the insect is a psyllid.
6. The method of claim 5 , wherein the psyllid is Diaphorina citri.
7. The method of claim 3 , wherein the method detects an HLB infection in a citrus grove selected from the group consisting of sweet orange, sour orange, mandarin, kumquat, tangerine, tangelo, lemon, lime, grapefruit, pumelo, Mexican lime, and combava.
8. The method of claim 3 , wherein the biological sample is selected from the group consisting of sweet orange, sour orange, mandarin, kumquat, tangerine, tangelo, lemon, lime, grapefruit, pumelo, Mexican lime, and combava.
9. The method of claim 3 , wherein amplicon is detected using a probe comprising:
(SEQ ID NO: 3)
5′ CACGCA-MeOG-C-MeOA-G-MeOA-C-MeOG-G-MeOG-MeOU-
G-MeOA-G-MeOU-A-MeOA-C-MeOG-TGCGTG 3′.
10. A method for detecting HLB in a plant or insect, the method comprising
extracting a nucleic acid molecule from a plant or insect,
contacting the extract with forward and reverse primers having the following sequences, respectively:
(SEQ ID NO: 1)
5′ GGACTCCATATGGAGTCCTCGCGAGCGG-MeOC-MeOA-MeOG-
MeOA-MeOC 3′
and
(SEQ ID NO: 2)
5′ TGACTCCATATGGAGTCATCTAGATTCC-MeOU-MeOA-MeOC-
MeOG-MeOC 3′
in the presence of a Nt.BstNBI(NEB) nicking enzyme, dNTPs, and a Bst DNA polymerase I for at least about ten minutes; and
detecting the presence or absence of a Liberibacter asiaticus amplicon, wherein the presence of a Liberibacter asiaticus amplicon identifies an HLB infection in the plant or insect, and the absence of a Liberibacter asiaticus amplicon identifies the absence of an HLB infection in the plant or insect.