Development of a droplet digital PCR method for detection of porcine circovirus 4

Abstract

Background Porcine circovirus 4 (PCV4), a newly emerging virus that was first discovered in 2019, may pose a potential threat to the pig industry. Droplet digital PCR (ddPCR) is an absolute quantitative method that has high sensitivity and accuracy. In this study, we developed a novel ddPCR assay to detect PCV4. Furthermore, we evaluated the detection limit, sensitivity, specificity and reproducibility of the ddPCR and TaqMan real-time quantitative PCR (qPCR) and tested 160 clinical samples to compare the detection rate of the two methods. Results The detection limit for ddPCR was 0.54 copies/µL, 10.6 times greater sensitivity than qPCR. Both ddPCR and qPCR assays exhibited good linearity and repeatability, and the established ddPCR method was highly specific for PCV4. The results of clinical sample testing showed that the positivity rate of ddPCR (5.6%) was higher than that of qPCR (4.4%). Conclusions This study successfully developed a sensitive, specific and repeatable ddPCR assay for PCV4 detection, which can be widely used in clinical diagnosis of PCV4 infections.

Droplet digital PCR aids in the diagnosis of children with fever of unknown origin ——A typical case report

“…In a study of bloodstream infections, ddPCR still had a sensitivity of 55.56 % and a specificity of 78.75 % in patients who had previously received antimicrobial therapy [ 24 ]. In addition, the sensitivity is higher than other nucleic acid detection methods such as qRT-PCR [ 5 ]. mNGS achieves wide coverage and has been widely used in patients with FUO.…”

“…A large portion of pediatric FUO causes (40 %–50 %) can be traced back to infections, emphasizing the need for thorough pathogen screening and the use of blood pathogen nucleic acid detection as advised by some guidelines [ 4 ]. However, despite their widespread use in clinical settings, quantitative real-time polymerase chain reaction (qRT-PCR) and metagenomic next-generation sequencing (mNGS) may miss the pathogens due to the low levels of nucleic acid in samples [ 5 ].…”

“…Compared to qRT-PCR and mNGS, droplet digital polymerase chain reaction (ddPCR) is a one-step amplification technique that has been proven to provide increased sensitivity in detecting nucleic acids [ 5 , 6 ]. Recently, ddPCR has shown promising potential in the fight against infections, but its application in FUO has not yet been adequately explored [ 7 ].…”