Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3)

Abstract

Koi herpesvirus disease (KHVD) is a highly infectious disease leading to outbreaks and mass mortality in captive and free-ranging common carp and koi carp. Outbreaks may result in high morbidity and mortality which can have a severe economic impact along the supply chain. Currently, control and prevention of KHVD relies on avoiding exposure to the virus based on efficient hygiene and biosecurity measures. An early diagnosis of the disease is crucial to prevent its spread and to minimize economic losses. Therefore, an easy-to-handle, sensitive, specific and reliable test prototype for a point-of-care detection of KHV was developed and evaluated in this study. We used a multiplex-endpoint-PCR followed by a specific probe hybridization step. PCR-products/hybridization-products were visualized with a simple and universal lateral flow immunoassay (PCR-LFA). Fifty-four gill tissue samples (KHV-positive n = 33, KHV-negative n = 21) and 46 kidney samples (KHV-positive n = 24, KHV-negative n = 22) were used to determine diagnostic sensitivity and specificity of the PCR-LFA. In addition, the usability of PCR-LFA to detect CyHV-3-DNA in gill swabs taken from 20 perished common carp during a KHVD-outbreak in a commercial carp stock was examined. This assay gave test results within approximately 60 min. It revealed a detection limit of 9 KHV gene copies/μl (95% probability), a diagnostic specificity of 100%, and diagnostic sensitivity of 94.81% if samples were tested in a single test run only. PCR inhibition was noticed when examining gill swab samples without preceding extraction of DNA or sample dilution. Test sensitivity coud be enhanced by examining samples in five replicates. Overall, our PCR-LFA proved to be a specific, easy-to-use and time-saving point-of-care-compatible test for the detection of KHV-DNA. Regarding gill swab samples, further test series using a higher number of clinical samples should be analyzed to confirm the number of replicates and the sample processing necessary to reveal a 100% diagnostic sensitivity.

Comparison of pre-labelled primers and nucleotides as DNA labelling method for lateral flow detection of Legionella pneumophila amplicons

“…It should be noted that this statement seems true for a simple singleplex assay. There are various studies that show that multiplex analyses using PCR-LFA are also technically possible [53][54][55] . Apart from the fact that the test strip design has to be extended for this, e.g.…”

Point‐of‐Care Testing (POCT) Devices for DNA Detection: A Comprehensive Review

“…In contrast, the NALFIA method is based on labeling DNA targets using low-molecular-weight tags, such as biotin, digoxigenin, and fluorescein. [34,[48][49][50][51] The sample preparation for this method includes amplifying target DNA or RNA with labeled primer. The sample is subsequently applied to LFA to form a complex with the capture ligand in the test zone.…”

Herpesviruses in Reptiles

“…Sensitive molecular-based rapid assays are relatively expensive and yet to be employed for the diagnosis of reptilian HVs (182)(183)(184)(185). We would propose an ultrasensitive format that combines PCR and immunoassay but then it can be argued that such a laboratory-based system is less rapid and has limited use in low-class laboratories (186,187). Rapid detection techniques such as Microfluidic chip immunoassay and Smartphone-based rapid telemonitoring system (SBRTS) are fast becoming powerful tools in the diagnosis of viral infections (188)(189)(190)(191)(192)(193)(194)(195)(196).…”