Multiplex assay for the detection of at least two citrus pathogens

Patent metadata

Field	Value
Publication number	US-2016145678-A1
Application number	US-201414905741-A
Country	US
Kind code	A1
Filing date	Jul 18, 2014
Priority date	Jul 19, 2013
Publication date	May 26, 2016
Grant date	—

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 a first citrus pathogen selected from a Huanglongbing, Witches' broom, Citrus Variegated Chlorosis (CVC), and citrus stubborn pathogen in a plant sample, the method comprising:
extracting DNA from said sample; performing a multiplex branched DNA signal amplification reaction; wherein the reaction comprises at least one capture extender probe and at least one label extender probe that targets the pathogen, wherein the at least one capture extender probe and the at least one label extender probe each comprises at least 8, 9, or 10 contiguous nucleotides of a probe sequence shown in Table 1; and detecting the presence or absence of a signal above background, wherein the presence of the signal is indicative of the presence of the pathogen in the plants sample.

2 . The method of claim 1 , wherein the reaction comprises multiple capture extender probes and multiple label extender probes that target the pathogen, wherein each of the multiple capture extender probes and multiple label extender probes that target the pathogen comprise at least 8, 9, or 10 contiguous nucleotides of a probe sequence as shown in Table 1.

3 . The method of claim 1 or claim 2 , wherein each probe in the reaction that targets the pathogen comprises a probe sequence set forth in Table 1.

4 . The method of claim 1 , wherein the reaction comprises: each capture extender probe, or a variant thereof, shown in Table 1 for the pathogen, wherein the variant comprises at least 8, 9, or 10 contiguous nucleotides of the capture extender probe sequence shown in Table 1; and/or each label extender probe, or a variant thereof, shown in Table 1 for the pathogen, wherein the variant comprises at least 8, 9, or 10 contiguous nucleotides of the label extender probe sequence shown in Table 1.

5 . The method of any one of claims 1 to 4 , wherein the reaction further comprises a blocking probe shown in Table 1 for the pathogen, or a variant thereof that comprises at least 8, 9, or 10 contiguous nucleotides of the blocking probe sequence as shown in Table 1.

6 . The method of claim 1 , further comprising detecting the presence or absence of a second citrus pathogen, different from the first citrus pathogen, selected from a Huanglongbing, Witches' broom, Citrus canker, CVC, and citrus stubborn pathogen in the plant sample, wherein the reaction comprises at least one capture extender probe and at least one label extender probe that targets the second pathogen wherein the capture extender probe and the label extender probe that target the second pathogen each comprise at least 8, 9, or 10 contiguous nucleotides of a probe sequence as shown in Table 1.

7 . The method of claim 6 , wherein the reaction comprises multiple capture extender probes and multiple label extender probes that target the second pathogen, wherein each of the multiple capture extender probes and multiple label extender probes that target the second pathogen comprise at least 8, 9, or 10 contiguous nucleotides of a probe sequence as shown in Table 1.

8 . The method of claim 6 or claim 7 , wherein each probe in the reaction that targets the second pathogen comprises a probe sequence as shown in Table 1.

9 . The method of claim 6 , wherein the reaction comprises: each capture extender probe, or a variant thereof, shown in Table 1 for the second pathogen, wherein the variant comprises at least 8, 9, or 10 contiguous nucleotides of the capture extender probe sequence shown in Table 1; and/or each label extender probe, or a variant thereof, shown in Table 1 for the second pathogen, wherein the variant comprises at least 8, 9, or 10 contiguous nucleotides of the label extender probe sequence shown in Table 1.

10 . The method of any one of claims 6 to 9 , wherein the reaction further comprises a blocking probe shown in Table 1 for the second pathogen, or a variant thereof that comprises at least 8, 9, or 10 contiguous nucleotides of a blocking probe sequence as shown in Table 1.

11 . The method of claim 6 , further comprising detecting the presence or absence of a third citrus pathogen, different from the first and second citrus pathogen, selected from a Huanglongbing, Witches' broom, Citrus canker, CVC, and citrus stubborn pathogen in the plant sample, wherein the reaction comprises at least one capture extender probe and at least one label extender probe that targets the third pathogen, wherein the capture extender probe and the label extender probe that target the third pathogen each comprise at least 8, 9, or 10 contiguous nucleotides of a probe sequence as shown in Table 1.

12 . The method of claim 11 , wherein the reaction comprises multiple capture extender probes and multiple label extender probes that target the third pathogen, wherein each of the multiple capture extender probes and multiple label extender probes that target the third pathogen comprise at least 8, 9, or 10 contiguous nucleotides of a probe sequence as shown in Table 1.

13 . The method of claim 11 or claim 12 , wherein each probe in the reaction that targets the third pathogen comprise a probe sequence as shown in Table 1.

14 . The method of claim 11 , wherein the reaction comprises: each capture extender probe, or a variant thereof, shown in Table 1 for the third pathogen, wherein the variant comprises at least 8, 9, or 10 contiguous nucleotides of the capture extender probe sequence shown in Table 1; and/or each label extender probe, or a variant thereof, shown in Table 1 for the third pathogen, wherein the variant comprises at least 8, 9, or 10 contiguous nucleotides of the label extender probe sequence shown in Table 1.

15 . The method of any one of claims 1 to 14 , wherein the reaction further comprises a blocking probe shown in Table 1 for the third pathogen, or a variant thereof that comprises at least 8, 9, or 10 contiguous nucleotides of a blocking probe sequence as shown in Table 1.

16 . The method of claim 11 , further comprising detecting the presence or absence of a fourth citrus pathogen, different from the first, second and third pathogen, selected from a Huanglongbing, Witches' broom, Citrus canker, CVC, and citrus stubborn pathogen in the plant sample, wherein the reaction comprises at least one capture extender probe and at least one label extender probe that targets the fourth pathogen wherein the capture extender probe and the label extender probe that target the fourth pathogen each comprise at least 8, 9, or 10 contiguous nucleotides of a probe sequence as shown in Table 1.

17 . The method of claim 16 , wherein the reaction comprises multiple capture extender probes and multiple label extender probes that target the fourth pathogen, wherein each of the multiple capture extender probes and multiple label extender probes that target the fourth pathogen comprise at least 8, 9, or 10 contiguous nucleotides of a probe sequence as shown in Table 1.

18 . The method of claim 16 or claim 17 , wherein each probe in the reaction that targets the fourth pathogen comprises a probe sequence as shown in Table 1.

19 . The method of claim 16 , wherein the reaction comprises: each capture extender probe, or a variant thereof, shown in Table 1 for the fourth pathogen, wherein the variant comprises at least 8, 9, or 10 contiguous nucleotides of the capture extender probe sequence shown in Table 1; and/or each label extender probe, or a variant thereof, shown in Table 1 for the fourth pathogen, wherein the variant comprises at least 8, 9, or 10 contiguous

Assignees

• Univ California

Inventors

• Wang Jinbo
• Dang Tyler
• Vidalakis Georgios

Classifications

• C12Q2600/158

  Expression markers · CPC title

• C12Q1/701

  Specific hybridization probes · CPC title

• C12Q2600/13

  Plant traits · CPC title

• C12Q1/689 Primary

  for bacteria · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title