Development and evaluation of rapid novel isothermal amplification assays for important veterinary pathogens: Chlamydiapsittaci and Chlamydiapecorum
Martina Jelocnik1, Md. Mominul Islam1, Danielle Madden1, Cheryl Jenkins2, James Branley3, Scott Carver4 and Adam Polkinghorne1
1Centre for Animal Health Innovation, University of the Sunshine Coast, Maroochydore, Queensland, Australia
2NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, NewSouth Wales, Australia
3Nepean Hospital, Penrith, NewSouthWales, Australia
4School of Biological Sciences, University of Tasmania, Hobart, Tasmania, Australia
Background Chlamydia psittaci and Chlamydia pecorum are important veterinary pathogens, with the former also being responsible for zoonoses, and the latter adversely affecting koala populations in Australia and livestock globally. The rapid detection of these organisms is still challenging, particularly at the point-of-care (POC). In the present study, we developed and evaluated rapid, sensitive and robust C. psittaci-specific and C. pecorum-specific Loop Mediated Isothermal Amplification (LAMP) assays for detection of these pathogens.
Methods and Materials The LAMP assays, performed in a Genie III real-time fluorometer, targeted a 263 bp region of the C. psittaci-specific Cps_0607 gene or a 209 bp region of a C.pecorum-specific conserved gene CpecG_0573, and were evaluated using a range of samples previously screened using species-specific quantitative PCRs (qPCRs). Species-specificity for C.psittaci and C.pecorum LAMP targets was tested against DNA samples from related chlamydial species and a range of other bacteria. In order to evaluate pathogen detection in clinical samples, C.psittaci LAMP was evaluated using a total of 26 DNA extracts from clinical samples from equine and avian hosts, while for C. pecorum LAMP, we tested a total of 63 DNA extracts from clinical samples from koala, sheep and cattle hosts. A subset of 36 C.pecorum samples was also tested in a thermal cycler (instead of a real-time fluorometer) using newly developed LAMP and results were determined as an end point detection. We also evaluated rapid swab processing (without DNA extraction) to assess the robustness of these assays.
Results Both LAMP assays were demonstrated to species-specific, highly reproducible and to be able to detect as little as 10 genome copy number/reaction, with a mean amplification time of 14 and 24 min for C. psittaci and C. pecorum, respectively. When testing clinical samples, the overall congruence between the newly developed LAMP assays and qPCR was 92.3% for C.psittaci (91.7% sensitivity and 92.9% specificity); and 84.1% for C.pecorum (90.6% sensitivity and 77.4% specificity). For a subset of 36 C.pecorum samples tested in a thermal cycler using newly developed LAMP, we observed 34/36 (94.4%) samples result being congruent between LAMP performed in fluorometer and in thermal cycler. Rapid swab processing method evaluated in this study also allows for chlamydial DNA detection using LAMP.
Discussion In this study, we describe the development of novel, rapid and robust C.psittaci-specific and C.pecorum-specific LAMP assays that are able to detect these bacteria in clinical samples in either the laboratory or POC settings. With further development and a focus on the preparation of these assays at the POC, it is anticipated that both tests may fill an important niche in the repertoire of ancillary diagnostic tools available to clinicians.