Multiplex assay for the detection of citrus pathogens

Patent metadata

Field	Value
Publication number	US-9347106-B2
Application number	US-201313945350-A
Country	US
Kind code	B2
Filing date	Jul 18, 2013
Priority date	Jul 18, 2012
Publication date	May 24, 2016
Grant date	May 24, 2016

Abstract

The present invention provides methods and compositions for detecting multiple citrus pathogens using a multiplex branched DNA signal amplification reaction.

First claim

What is claimed is:

1. A method for detecting the presence or absence of nine citrus pathogens Citrus tristeza virus (CTV) universal, CTV genotype T30, CTV genotype VT, Citrus psorosis virus, Citrus tatter leaf virus, Citrus leaf blotch virus, Citrus exocortis viroid, Hop stunt viroid, and Citrus leprosis virus in a plant sample, the method comprising:
extracting RNA from said sample;
performing a multiplex branched DNA signal amplification reaction; wherein the reaction comprises a probe set that targets Citrus tristeza virus (CTV) universal, a probe set that targets CTV genotype T30, a probe set that targets CTV genotype VT, a probe set that targets Citrus psorosis virus, a probe set that targets Citrus tatter leaf virus, a probe set that targets Citrus leaf blotch virus, a probe set that targets Citrus exocortis viroid, a probe set that targets Hop stunt viroid, and a probe set that targets Citrus leprosis virus, wherein each probe set comprises at least one capture extender probe and a at least one label extender probe, and wherein;
the at least one capture extender probe that targets CTV universal comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:1, 2, 3, 4, 5, or 6; and the at least one label extender probe that targets CTV universal comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO: 7, 8, 9, 10, 11, or 12;
the at least one capture extender probe that targets CTV genotype T30 comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:13, 14, 15, 16, 17, 18, 19, or 20; and the at least one label extender probe that targets CTV genotype T30 comprises at least 8, 9, or 10 contiguous of a probe sequence of SEQ ID NO:21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38;
the at least one capture extender probe that targets CTV VT genotype comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:41, 42, 43, 44, 45, or 46; and the at least one label extender probe that targets CTV VT genotype comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or 66;
the at least one capture extender probe that targets Citrus psorosis virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:71, 72, 73, 74, 75, 76, 77, or 78; and the at least one label extender probe that targets Citrus psorosis virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:79, 80, 81, 82, 83, 84, 85, or 86;
the at least one capture extender probe that targets Citrus tatter leaf virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:87, 88, 89, 90, 91, or 92; and the at least one label extender probe that targets Citrus tatter leaf virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:93, 94, 95, 96, 97, 98, or 99;
the at least one capture extender probe that targets Citrus leaf blotch virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:100, 101, 102, 103, or 104; and the at least one label extender probe that targets Citrus leaf blotch virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:105, 106, 107, 108, 109, 110, 111, 112, 113, or 114;
the at least one capture extender probe that targets Citrus exocortis virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:118, 119, 120, 121, 122, or 123; and the at least one label extender probe that targets Citrus exocortis virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:124, 125, 126, 127, 128, 129, 130, or 131;
the at least one capture extender probe that targets Hop stunt viroid comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:133, 134, 135, 136, or 137; and the at least one label extender probe that targets Hop stunt viroid comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:138, 139, 140, 141, 142, or 143;
the at least one capture extender probe that targets Citrus leprosis virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:144, 145, 146, 147, 148, or 149; and the at least one label extender probe that targets Citrus leprosis virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, or 161; and

detecting the presence or absence of a signal above background from at least one of the probe sets, wherein the presence of the signal is indicative of the presence of the pathogen that the probe set targets.

2. The method of claim 1 , wherein the reaction comprises multiple capture extender probes and multiple label extender probes that target each of the nine pathogens, wherein:
each of the multiple capture extender probes that target CTV universal comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:1, 2, 3, 4, 5, or 6; and each of the label extender probes that target CTV universal comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO: 7, 8, 9, 10, 11, or 12;
each of the multiple capture extender probes that target CTV genotype T30 comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:13, 14, 15, 16, 17, 18, 19, or 20; and each of the multiple extender probes that target CTV genotype T30 comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38;
each of the multiple capture extender probes that target CTV VT genotype comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:41, 42, 43, 44, 45, or 46; and each of the multiple label extender probes that target CTV VT genotype comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or 66;
each of the multiple capture extender probes that target Citrus psorosis virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:71, 72, 73, 74, 75, 76, 77, or 78; and each of the multiple extender probes that target Citrus psorosis virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:79, 80, 81, 82, 83, 84, 85, or 86;
each of the multiple capture extender probes that target Citrus tatter leaf virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:87, 88, 89, 90, 91, or 92; and each of the multiple label extender probes that target Citrus tatter leaf virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:93, 94, 95, 96, 97, 98, or 99;
each of the multiple capture extender probes that target Citrus leaf blotch virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:100, 101, 102, 103, or 104; and each of the multiple label extender probes that target Citrus leaf blotch virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:105, 106, 107, 108, 109, 110, 111, 112, 113, or 114;
each of the multiple capture extender probes that target Citrus exocortis virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:118, 119, 120, 121, 122, or 123; and each of the multiple label extender probes that target Citrus exocortis virus comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence of SEQ ID NO:124, 125, 126, 127, 128, 129, 130, or 131;
each of the multiple capture extender probe

Assignees

• Univ California

Inventors

• Wang Jinbo

Classifications

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q1/701 Primary

  Specific hybridization probes · CPC title