Loop-Mediated AMPlification (LAMP) Primers that Specifically Detect Clavibacter michiganensis subsp. nebraskensis

Case ID: 01025

Description

Corn is an important crop worldwide and the Goss’s wilt pathogen, Clavibacter michiganensis subspecies nebraskensis, can cause considerable losses.  Current diagnosis of Goss’s wilt is based on symptomology and isolation of C. michiganensis subsp. nebraskensis on semiselective medium.  Current identification of C. michiganensis subsp. nebraskensis is inaccurate and/or inefficient.  Loop-mediated amplification (LAMP) was tested using known subspecies of C. michiganensis subsp. nebraskensis strains and several known non-target bacteria, including all known subspecies of C. michiganensis.  The LAMP assay was also used to test bacterial isolates from diseased corn.  A combination of 16S rDNA and dnaA sequence analyses was used to confirm the identity of the isolates and only isolates determined to be C. michiganensis subsp. nebraskensis reacted positively with the LAMP assay, confirming specificity.  The Cmm ImmunoStrip assay was included as a presumptive identification test, as cross-reactions with C. michiganensis subsp. nebraskensis are known.  The LAMP assay was run in a handheld real-time monitoring device and performed equally to in-lab equipment.  This LAMP assay could become the standard identification assay for C. michiganensis subsp. nebraskensis and has potential as a field test, and the targeted gene has application across all molecular detection platforms.  These primers allow end-point and real-time assessment.  This diagnostic tool will provide an easy one-step test for rapid identification of Cmn.

 

Applications

These primers were designed for LAMP detection of Cmn and can be applied to corn field testing and corn seed testing. The LAMP assay can be used in-field with the non-instrumented nucleic acid amplification (NINA) device for end-point assessment and/or hand-held real-time assessment devices such as SMART-DART technology.

 

 

Advantages

These primers eliminate false results and allow for highly specific molecular detection of Cmn. These primers have been designed for LAMP detection. The nature of the LAMP assay itself is advantageous in that it is highly specific, rapid, results can be assessed at end-point or in real-time, the polymerase used is not affected by as many inhibitors as PCR, and the isothermal nature of the reaction eliminates the need for expensive thermo cycling equipment.

 

Patent Information:
Inventors
Jarred Yasuhara-Bell
Anne Alvarez

For information, contact:
Ann Park
Technology Licensing Associate
University of Hawaii
(808) 956-9929
apark@hawaii.edu
Keywords


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