Development and comparative evaluation of droplet digital PCR and quantitative PCR for the detection and quantification of Chlamydia psittaci

Sahu, Radhakrishna; Vishnuraj, M.R.; Srinivas, Ch; Dadimi, Bhargavi; Megha, G.K.; Pollumahanti, Niveditha; Malik, Satyaveer S.; Vaithiyanathan, S.; Rawool, Deepak B.; Barbuddhe, Sukhadeo B.

Development and comparative evaluation of droplet digital PCR and quantitative PCR for the detection and quantification of Chlamydia psittaci

2021

Chlamydia psittaci is a zoonotic pathogen mainly transmitted by psittacine birds and poultry. The low shedding rate of the pathogen in the apparently healthy birds and human clinical cases may result in false-negative results. In the present study, a droplet digital PCR (ddPCR) assay was developed and compared with optimized quantitative PCR (qPCR) for the detection of C. psittaci from the clinical samples. The ddPCR assay was found to be comparatively more sensitive than the qPCR, wherein the limit of detection (LOD) of ddPCR was upto 2.4 copies of the DNA template, whereas, the qPCR could detect upto 38 copies of the DNA template in the reaction mixture. Overall, the developed ddPCR assay was found to be robust, specific, and could reliably quantify up to 17.8 copies of the DNA template. Finally, the applicability of the developed ddPCR assay was tested by screening the field samples (n = 124), comprising lung tissues from dead poultry and feral birds; pooled faecal samples from the free-living birds, commercial and backyard poultry farms; pharyngeal and cloacal swabs collected from the duck farms. Of these, a total of seven samples were found to be positive by the ddPCR, whereas, three samples could be detected as positive using the qPCR. The developed ddPCR could serve as a reliable screening tool, particularly in those clinical samples wherein the shedding of C. psittaci is substantially very low.

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