Development and Clinical Validation of a Potential Penside Colorimetric Loop-Mediated Isothermal Amplification Assay of Porcine Circovirus Type 3

Abstract

Porcine circovirus type 3 (PCV3), a novel circovirus, imposes great burdens on theglobal pig industry. The penside tests for detecting PCV3 are critical for assessingthe epidemiological status and working out disease prevention and control programsdue to the unavailability of a commercial vaccine. A one-step molecular assay basedon visual loop-mediated isothermal amplification (vLAMP) was developed for simpleand rapid detection of PCV3. We compared its sensitivity and specificity with TaqManquantitative real-time polymerase chain reaction (qPCR) and applied the developedassay in the epidemiological study of (n = 407) pooled swine sera collected fromalmost the entire mainland China during the years 2017–2018. We also explored thefeasibility of the vLAMP assay for detecting raw samples without a prior DNA isolationstep to expand its application capability. Results showed that the vLAMP assay couldreliably detect the PCV3 cap gene with a detection limit of 10 DNA copies equalto that of the Taqman qPCR assay. In the epidemiological study, the PCV3 positivedetection rate for 407 swine pooled sera detected by the vLAMP assay was 37.35%(152/407), whereas it was 39.01% (159/407) for Taqman qPCR. For the detectionmethod without genome extraction, the results kept satisfactory specificity (100%) butdisplayed lower sensitivity (100% for CT < 32), indicating the direct detection is notsensitive enough to discriminate the samples with low viral loads. The one-step vLAMPis a convenient, rapid, and cost-effective diagnostic for penside detection and will enablethe epidemiological surveillance of PCV3, which has widely spread in mainland China