Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care

Abstract

Background C. psittaci has recently emerged as an equine abortigenic pathogen causing significant losses to the Australian Thoroughbred industry, while Equine herpesvirus-1 (EHV-1) is a well-recognized abortigenic agent. Diagnosis of these agents is based on molecular assays in diagnostic laboratories. In this study, we validated C. psittaci and newly developed EHV-1 Loop Mediated Isothermal Amplification (LAMP) assays performed in a real-time fluorometer (rtLAMP) against the reference diagnostic assays. We also evaluated isothermal amplification using commercially available colorimetric mix (cLAMP), and SYBR Green DNA binding dye (sgLAMP) for “naked eye” end-point detection when testing ‘real-world’ clinical samples. Finally, we applied the C. psittaci LAMP assays in two pilot Point-of-Care (POC) studies in an equine hospital. Results The analytical sensitivity of C. psittaci and EHV-1 rt-, and colorimetric LAMPs was determined as one and 10 genome equivalents per reaction, respectively. Compared to reference diagnostic qPCR assays, the C. psittaci rtLAMP showed sensitivity of 100%, specificity of 97.5, and 98.86% agreement, while EHV-1 rtLAMP showed 86.96% sensitivity, 100% specificity, and 91.43% agreement. When testing rapidly processed clinical samples, all three C. psittaci rt-, c-, sg-LAMP assays were highly congruent with each other, with Kappa values of 0. 906 for sgLAMP and 0. 821 for cLAMP when compared to rtLAMP. EHV-1 testing also revealed high congruence between the assays, with Kappa values of 0.784 for cLAMP and 0.638 for sgLAMP when compared to rtLAMP. The congruence between LAMP assays and the C. psittaci or EHV-1 qPCR assays was high, with agreements ranging from 94.12 to 100% for C. psittaci, and 88.24 to 94.12% for EHV-1, respectively. At the POC, the C. psittaci rt- and c-LAMP assays using rapidly processed swabs were performed by technicians with no prior molecular experience, and the overall congruence between the POC C. psittaci LAMPs and the qPCR assays ranged between 90.91–100%. Conclusions This study describes reliable POC options for the detection of the equine pathogens: C. psittaci and EHV-1. Testing ‘real-world’ samples in equine clinical setting, represents a proof-of-concept that POC isothermal diagnostics can be applied to rapid disease screening in the equine industry.

Real-Time Fluorometric Isothermal LAMP Assay for Detection of Chlamydia pecorum in Rapidly Processed Ovine Abortion Samples: A Veterinary Practitioner’s Perspective

“…In practice, detection of C. pecorum requires sending samples to a specialised veterinary laboratory where nucleic acid testing, such as qPCR, can be performed. It is evident from this pilot study and others [14,16,17] that LAMP has the potential to be applied as a rapid, POC diagnostic tool for veterinarians performing various disease investigations in livestock species. This has numerous benefits in a veterinary setting including the ability to quickly provide results to farmers and direct disease management strategies in shorter timeframes, and with lower cost compared to other molecular methods.…”

“…Nevertheless, the utility of these assays at clinical setting and point-of-care (POC) is evident in koala clinical practice, where several wildlife hospitals utilise the C. pecorum LAMP assays as the diagnostic toolkit [ 12 , 13 ] (personal communication Dr Amy Robbins, Endeavour Veterinary Ecology, and Dr Amber Gillet, Australia Zoo Wildlife Hospital). The utility of chlamydial rapid isothermal testing and rapid sample processing has also been demonstrated in equine clinical practice to detect C. psittaci in equine abortions, both at POC and in the diagnostic laboratories [ 14 ].…”

“…Considering the emerging evidence for the abortigenic potential of C. pecorum infections in sheep [ 8 , 9 , 15 ], a rapid diagnosis would offer effective on-farm surveillance, improve biosecurity, and aid in infection control [ 14 ]. While the rapid swab processing and isothermal testing has been evaluated for koala C. pecorum detection, there is a paucity of reports for the use of these in diagnosing C. pecorum infections in sheep.…”

One clone to rule them all: Culture-independent genomics of Chlamydia psittaci from equine and avian hosts in Australia

“…These swab samples were processed by adding 300 µl of sterile TE (Tris-EDTA) buffer, vortexing, and heat lysis at 95 °C for 10 min, followed by DNA extraction using the QIAmp DNA mini kit (Qiagen, Australia), as previously described [15]. Before WGS, all DNA samples were once again screened with C. psittaci qPCR to estimate genome copy number [15,18] (Supplementary Materials, Table S1), followed by DNA concentration and purity analyses using Qubit 3 Fluorometer and NanoDrop 1000 Spectrophotometer (both by ThermoFisher Scientific, Australia), respectively.…”

Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii

“…Therefore, isothermal amplification assays showed great potential in scientific and clinical research [ 26 ]. Loop-mediated isothermal amplification (LAMP) is one of the well-recognized isothermal amplification techniques applied in clinical diagnosis [ 27 , 28 ]. But LAMP needs specific design of 2 to 3 pairs of primers and the processes of primer design and selection are tedious [ 29 , 30 ].…”