Recipient Organization
VARIGEN BIOSCIENCES CORPORATION
505 S ROSA RD STE 15
MADISON,WI 537191262
Performing Department
(N/A)
Non Technical Summary
Objective of this project is to develop a diagnostic assay for rapid detection of avian metapneumovirus (aMPV) in less than 30 minutes using a novel multiplex molecular diagnostic assay.This diagnostic assay will be based on a multiplex loop-mediated isothermal amplification (mLAMP) method, which allows real-time detection of multiple targets in a single reaction. This diagnostic test will be designed to differentiate between common subtypes of aMPV (A, B, and C) in a single reaction. This assay will be performed on a isothermal instrument, and the results will be displayed on the screen as positive, negative, or non-conclusive.Assay performance will be compared with that of the reference method (real time RT-qPCR). A simple and rapid sample preparation method will also be developed. The analytical performance of the aMPV mLAMP assay will be evaluated by testing a cohort ofpositive (aMPV A/B/C) andnegative samples.
Animal Health Component
30%
Research Effort Categories
Basic
5%
Applied
30%
Developmental
65%
Goals / Objectives
The major goal of this project is to develop a diagnostic assay for rapid detection of avain metapenumovirus (aMPV) at point of care. Results of this assay will be available within 30 minutes.This diagnostic test will be designed to differentiate between common subtypes of aMPV (A, B, and C) in a single reaction.
Project Methods
Aim 1 - Designing of LAMP primers. LAMP primers specific for the detection of aMPV-B, aMPV-C, and avian 18S rRNA (for use as an internal positive control) will be designed.Aim 2 - Performance of primers in monoplex LAMP assay. Primers will be tested in a monoplex LAMP assay.Reaction conditions will be optimized for target amplification in less than 30 min. The sensitivity and specificity of the RT-LAMP assay will be determined and compared to a reference method.Aim 3 - Development of mLAMP assay. Primer sets selected fromAim 2 will be used to develop mLAMP assay. Various parameters (primer concentration, enzyme concentration, reaction temperature, etc.) will be optimized to ensure maximum sensitivity of the assay and no non-specific amplification. The sensitivity and specificity of the assay will be determined and compared with those of the reference method.Aim 4 - Sample preparation method development. A simple and easy to use sample preparation method that is compatible with the goals of a simple single-tube format with minimal steps and reagents that can be performed without the need for any equipment. Success will be measured by equivalent clinical sensitivity and specificity to standard extraction methodology.Aim 5 - Testing of clinical samples using mLAMP assay. To validate the clinical performance of mLAMP assay, a set of positive and negative clinical samples will be tested. Samples will be tested in a blinded manner, and the results obtained will be compared with the reference method to determine the clinical sensitivity/specificity of the test.