Molecular Detection of Infectious Laryngotracheitis Virus in Chickens with a Microfluidic Chip

SIMPLE SUMMARY: Infectious laryngotracheitis (ILT) presents a major risk to the chicken industry. Rapid, specific, simple, and point-of-need molecular detection of the virus is crucial to enable chicken farms to take timely action and contain the spread of infection. The current study describes an isothermal amplification assay for infectious laryngotracheitis virus (ILTV) infection and the implementation of this assay in a microfluidic chip suitable for molecular detection and quasi-quantification of ILTV in diagnostic veterinary laboratories with low resources and poultry farms. Our assay performance was compared and favorably agreed with quantitative PCR (qPCR). Clinical tests of our assay and chip with samples from diseased chickens demonstrated good concordance with the gold-standard benchtop qPCR assay. ABSTRACT: Infectious laryngotracheitis (ILT) is a viral disease of chickens’ respiratory system that imposes considerable financial burdens on the chicken industry. Rapid, simple, and specific detection of this virus is crucial to enable proper control measures. Polymerase chain reaction (PCR)-based molecular tests require relatively expensive instruments and skilled personnel, confining their application to centralized laboratories. To enable chicken farms to take timely action and contain the spread of infection, we describe a rapid, simple, semi-quantitative benchtop isothermal amplification (LAMP) assay, and a field-deployable microfluidic device for the diagnosis of ILTV infection in chickens. Our assay performance was compared and favorably agreed with quantitative PCR (qPCR). The sensitivity of our real-time LAMP test is 250 genomic copies/reaction. Clinical performance of our microfluidic device using samples from diseased chickens showed 100% specificity and 100% sensitivity in comparison with benchtop LAMP assay and the gold-standard qPCR. Our method facilitates simple, specific, and rapid molecular ILTV detection in low-resource veterinary diagnostic laboratories and can be used for field molecular diagnosis of suspected ILT cases.